Vanillin

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavour and aroma molecule, but is also of interest because of its biogenetic relationship to the phenylpropanoid pathway and to other molecules of physiological significance, notably salicylate. Recent progress towards characterisation of the biosynthesis of vanillin is reviewed. In Vanilla, there is some evidence that the route to vanillin-beta-D-glucoside may proceed from 4-coumaric acid via 4-hydroxybenzaldehyde, with glucoside formation occurring not necessarily as the final step, and possibly with the involvement of 4-hydroxybenzyl alcohol beta-D-glucoside tartrate bis-esters as "shunt" metabolites. This appears to be given tentative support by the recent partial characterisation of a 4-hydroxybenzaldehyde synthase from Vanilla. On the other hand, a well-characterised, CoA-dependent, non-oxidative chain-shortening mechanism to produce vanillin from ferulic acid, occurring as part of a pathway of hydroxycinnamate degradation in Pseudomonas, may not be representative of hydroxycinnamate chain-shortening mechanism(s) occurring in Vanilla and other plants. Nevertheless, by expression of the Pseudomonas enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), attempts have been made to introduce a direct capacity for vanillin formation into model plants by diversion of the phenylpropanoid pathway. The results obtained have emphasised the obstacles to achieving the desired oxidation level (aldehyde) and ring substitution (4-hydroxy-3-methoxyphenyl), even when a substantial metabolic diversion is successfully achieved. Finally, the significance of the latest biosynthetic and biotechnological developments is reviewed briefly in relation to authentication of vanillin.     

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.     

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.     

De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae)

Vanillin is one of the world''s most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world''s vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker''s yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.In 2007, the global market for flavor and fragrance compounds was an impressive $20 billion, with an annual growth of 11 to 12%. The isolation and naming of vanillin (3-methoxy-4-hydroxybenzaldehyde) as the main component of vanilla flavor in 1859 (8), and the ensuing chemical synthesis in 1874 (41), in many ways marked the true birth of this industry, and this compound remains the global leader in aroma compounds. The original source of vanillin is the seed pod of the vanilla orchid (Vanilla planifolia), which was grown by the Aztecs in Mexico and brought to Europe by the Spaniards in 1520. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1- to 6-month curing process of the harvested green vanilla pods (37). Production of 1 kg of vanillin requires approximately 500 kg of vanilla pods, corresponding to the pollination of approximately 40,000 flowers. Today, only about 0.25% (40 tons out of 16,000) of vanillin sold annually originates from vanilla pods, while most of the remainder is synthesized chemically from lignin or fossil hydrocarbons, in particular guaiacol. Synthetically produced vanillin is sold for approximately $15 per kg, compared to prices of $1,200 to $4,000 per kg for natural vanillin (46).An attractive alternative is bioconversion or de novo biosynthesis of vanillin; for example, vanillin produced by microbial conversion of the plant constituent ferulic acid is marketed at $700 per kilogram under the trade name Rhovanil Natural (produced by Rhodia Organics). Ferulic acid and eugenol are the most attractive plant secondary metabolites amenable for bioconversion into vanillin, since they can be produced at relatively low costs: around $5 per kilogram (37). For the bioconversion of eugenol or ferulic acid into vanillin, several microbial species have been tested, including gram-negative bacteria of the Pseudomonas genus, actinomycetes of the genera Amycolatopsis and Streptomyces, and the basidiomycete fungus Pycnoporus cinnabarinus (19, 23, 25, 27, 31, 34, 35, 36, 45, 48). In experiments where the vanillin produced was absorbed on resins, Streptomyces cultures afforded very high vanillin yields (up to 19.2 g/liter) and conversion rates as high as 55% were obtained (15). Genes for the responsible enzymes from some of these organisms were isolated and expressed in Escherichia coli, and up to 2.9 g/liter of vanillin were obtained by conversion of eugenol or ferulic acid (1, 3, 32, 49).Compared to bioconversion, de novo biosynthesis of vanillin from a primary metabolite like glucose is much more attractive, since glucose costs less than $0.30/kilogram (42). One route for microbial production of vanillin from glucose was devised by Frost and coworker Li (6, 20), combining de novo biosynthesis of vanillic acid in E. coli with enzymatic in vitro conversion of vanillic acid to vanillin. 3-Dehydroshikimic acid is an intermediate in the shikimate pathway for biosynthesis of aromatic amino acids, and the recombinant E. coli was engineered to dehydrate this compound to form protocatechuic acid (3,4-dihydroxybenzoic acid) and methylate this to form vanillic acid. The vanillic acid was subsequently converted into vanillin in vitro using carboxylic acid reductase isolated from Neurospora crassa. The main products of the in vivo step were protocatechuic acid, vanillic acid, and isovanillic acid in an approximate ratio of 9:4:1, indicating a bottleneck at the methylation reaction and nonspecificity of the OMT (O-methyltransferase) enzyme for the meta-hydroxyl group of protocatechuic acid. Serious drawbacks of this scheme are the lack of an in vivo step for the enzymatic reduction of vanillic acid, demanding the addition of isolated carboxylic acid reductase and costly cofactors such as ATP, NADPH, and Mg²⁺, and the generation of isovanillin as a contaminating side product.In this study, we have genetically engineered single-recombination microorganisms to synthesize vanillin from glucose, according to the metabolic route depicted in Fig. ​Fig.1.1. To avoid the synthesis of isovanillin as an undesired side product, a large array of OMTs was screened for the desired high substrate specificity, and an appropriate enzyme was identified. A synthetic version of an aromatic carboxylic acid reductase (ACAR) gene, optimized for yeast codon usage, was introduced to achieve the reduction step. The vanillin pathway was introduced into both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, and significant levels of vanillin production were obtained in both organisms. Vanillin β-d-glucoside is the form in which vanillin accumulates and is stored in the fresh pod of the vanilla orchid (Vanilla planifolia). During the “curing” process of the pod, β-glucosidases are liberated and facilitate a partial conversion of the vanillin β-d-glucoside into vanillin. Upon consumption or application, the conversion of vanillin β-d-glucoside into free vanillin by enzymes in the saliva or in the skin microflora can provide for a slow-release effect that prolongs and augments the sensory event, as is the case for other flavor glycosides investigated, such as menthol glucoside (14, 16). In addition to the increased value of vanillin β-d-glucoside as an aroma or flavor compound, production of the glucoside in yeast may offer several advantages. Vanillin β-d-glucoside is more water soluble than vanillin, but most importantly, compounds such as vanillin in high concentrations are toxic to many living cells (4). It has been shown that glucosides of toxic compounds are less toxic to yeasts (24). We found this to be the case with vanillin and S. cerevisiae yeast as well. Thus, to facilitate storage and accumulation of higher vanillin yields, we introduced a step for vanillin glucosylation in S. pombe.Open in a separate windowFIG. 1.Biosynthetic scheme for de novo biosynthesis of vanillin in Schizosaccharomyces pombe and outline of the different vanillin catabolites and metabolic side products observed in different yeast strains and constructs. Gray arrows, primary metabolic reactions in yeast; black arrows, enzyme reactions introduced by metabolic engineering; diagonally striped arrows, undesired inherent yeast metabolic reactions.     

Accumulation of cell wall-bound phenolic metabolites and their upliftment in hairy root cultures of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

Alkaline hydrolysis of cell wall material of tomato hairy roots yielded ferulic acid as the major phenolic compound. Other phenolics were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. The content of phenolics was much higher at the early stage of hairy root growth. The ferulic acid content decreased up to 30 days and then sharply increased to 360 microg/g at 60 days of growth. Elicitation of hairy root cultures with Fusarium mat extract (FME) increased ferulic acid content 4-fold after 24 h. As the pathogen-derived elicitors have specific receptors in plants, FME may thus be used for inducing resistance against Fusarium oxysporum f. sp. lycopersici.     

Characterization of a multifunctional methyltransferase from the orchid <Emphasis Type="Italic">Vanilla planifolia</Emphasis>

The final enzymatic step in the synthesis of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of 3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA that encodes a multifunctional methyltransferase from Vanilla planifolia tissue cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The higher catalytic efficiency of the purified recombinant enzyme with the substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue distribution, suggest this methyltransferase may primarily function in lignin biosynthesis. However, since the enzyme characterized here does have 3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in engineering strategies for the synthesis of natural vanillin from alternate sources.Abbreviations COMT Caffeic acid O-methyltransferase - DOMT 3,4-Dihydroxybenzaldehyde-O-methyltransferase - OMTs O-Methyltransferases - SAM S-adenosyl-l-methionine     

Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus

The inhibitory effects of various lignocellulose degradation products on glucose fermentation by the thermotolerant yeast Kluyveromyces marxianus were studied in batch cultures. The toxicity of the aromatic alcohol catechol and two aromatic aldehydes (4-hydroxybenzaldehyde and vanillin) was investigated in binary combinations. The aldehyde furfural that usually is present in relatively high concentration in hydrolyzates from pentose degradation was also tested. Experiments were conducted by combining agents at concentrations that individually caused 25% inhibition of growth. Compared to the relative toxicity of the individual compounds, combinations of furfural with catechol and 4-hydroxybenzaldehyde were additive (50% inhibition of growth). The other binary combinations assayed (catechol with 4-hydroxybenzaldehyde, and vanillin with catechol, furfural, or 4-hydroxybenzaldehyde) showed synergistic effect on toxicity and caused a 60-90% decrease in cell mass production. The presence of aldehydes in the fermentation medium strongly inhibited cell growth and ethanol production. Kluyveromyces marxianus reduces aldehydes to their corresponding alcohols to mitigate the toxicity of these compounds. The total reduction of aldehydes was needed to start ethanol production. Vanillin, in binary combination, was dramatically toxic and was the only compound for which inhibition could not be overcome by yeast strain assimilation, causing a 90% reduction in both cell growth and fermentation.     

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.     

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.     

Studies on the Flavors in Saké

A very small amount of vanillin was found in Saké, but the mechanism of its formation during Saké brewing has not yet been elucidated. Therefore, shaking culture of a Saké yeast (Kyokai No. 7 strain) was carried out in the Hayduck’s solution containing ferulic acid which was considered to be a precursor of vanillin. By the analysis of the fermentation products, formation of p-hydroxybenzoic acid and vanillic acid was elucidated. On the other hand, in the similar experiment using vanillin in place of ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid were identified.

On these results, it was suggested that vanillin might be formed as an intermediate of the degradation reaction of ferulic acid, and also, the demethoxylation of vanillin might be occurred in the fermentation of yeast.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.     

Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF

Steam-exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam-explosion pretreatment process. The SE temperature and time conditions were 214 degrees C and 6 min, resulting in a log R(0) of 4.13. In enzymatic hydrolysis tests at 45 degrees C, the biomass loading in the bioreactor was 100 g(DW)/L (dry weight) and the enzyme-to-biomass ratio 0.06 g/g(DW). The enzyme activities for endo-glucanase, exo-glucanase, and beta-glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5-hydroxymethyl furfural (5-HMF), syringaldehyde, 4-hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5-HMF, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5-HMF (0.5 g/L) in SSF of 100 g(DW) /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.     

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.     

Studies on the Flavors of Roast Barley (Mugi-Cha)

Five weakly acidic carbonyl compounds and two neutral carbonyl compounds were newly isolated as their 2,4-dinitrophenylhydrazones besides the previously reported compounds by using column and thin-layer chromatographies.

These compounds were characterized and identified by their IR, UV and MS spectra and the mixed melting points test.

The newly isolated and identified compounds were as follows; weakly acidic carbonyl compounds: 2-pyrrolealdehyde, vanillin, p-hydroxybenzaldehyde, syringaldehyde, proto-catechuic aldehyde, neutral carbonyl compounds: glyoxal, 5-hydroxymethylfurfural.

These compounds, particularly vanillin, protocatechuic aldehyde, 2-pyrrolealdehyde and 5-hydroxymethylfurfural, appeared to be concerned with the flavor of roast barley (Mugi-Cha).     

Enhancement of retronasal odors by taste

Psychophysical studies of interactions between retronasal olfaction and taste have focused most often on the enhancement of tastes by odors, which has been attributed primarily to a response bias (i.e., halo dumping). Based upon preliminary evidence that retronasal odors could also be enhanced by taste, the present study measured both forms of enhancement using appropriate response categories. In the first experiment, subjects rated taste ("sweet," "sour," "salty," and "bitter") and odor ("other") intensity for aqueous samples of 3 tastants (sucrose, NaCl, and citric acid) and 3 odorants (vanillin, citral, and furaneol), both alone and in taste-odor mixtures. The results showed that sucrose, but not the other taste stimuli, significantly increased the perceived intensity of all 3 odors. Enhancement of tastes by odors was inconsistent and generally weaker than enhancement of odors by sucrose. A second experiment used a flavored beverage and a custard dessert to test whether the findings from the first experiment would hold for the perception of actual foods. Adding sucrose significantly enhanced the intensity of "cherry" and "vanilla" flavors, whereas adding vanillin did not significantly enhance the intensity of sweetness. It is proposed that enhancement of retronasal odors by a sweet stimulus results from an adaptive sensory mechanism that serves to increase the salience of the flavor of nutritive foods.     

Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics

The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.     

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.