The isolation and identification of Bacillus velezensis ZN-S10 from vanilla (V. planifolia), and the microbial distribution after the curing process.

The market value of vanilla beans (Vanilla planifolia) is constantly increasing due to their natural aroma and flavor properties that improve after a curing process, where bacteria colonization plays a critical role. However, a few publications suggest that bacteria play a role in the curing process. Hence, this study aimed to isolate Bacillus sp. that could be used for fermenting V. planifolia while analyzing their role in the curing process. Bacillus velezensis ZN-S10 identified with 16S rRNA sequencing was isolated from conventionally cured V. planifolia beans. A bacteria culture solution of B. velezensis ZN-S10 (1 mL of 1 × 107 CFU mL-1) was then coated on 1 kg of non-cured vanilla pods that was found to ferment and colonize vanilla. PCA results revealed distinguished bacterial communities of fermented vanilla and the control group, suggesting colonization of vanilla. Phylogenetic analysis showed that ZN-S10 was the dominant Bacillus genus member and narrowly correlated to B. velezensis EM-1 and B. velezensis PMC206-1, with 78% and 73% similarity, respectively. The bacterial taxonomic profiling of cured V. planifolia had a significant relative abundance of Firmicutes, Proteobacteria, Cyanobacteria, Planctomycetes, and Bacteroidetes phyla according to the predominance. Firmicutes accounted for 55% of the total bacterial sequences, suggesting their colonization and effective fermentation roles in curing vanilla.

Vanilla from Brazilian Atlantic Forest: In vitro and in silico toxicity assessment and high-resolution metabolomic analysis of Vanilla spp. ethanolic extracts.

The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and susceptibility to pathogens. There is an increasing demand for natural vanilla, prized for its complex, authentic, and superior quality compared to artificial counterparts. Therefore, there is a necessity for innovative production alternatives to ensure a consistent and stable supply of vanilla flavors. In this context, vanilla crop wild relatives (WRs) emerge as promising natural sources of the spice. However, these novel species must undergo toxicity assessments to evaluate potential risks and ensure safety for consumption. This study aimed to assess the non-mutagenic and non-carcinogenic properties of ethanolic extracts from V. bahiana, V. chamissonis, V. cribbiana, and V. planifolia through integrated metabolomic profiling, in vitro toxicity assays, and in silico analyses. The integrated approach of metabolomics, in vitro assays, and in silico analyses has highlighted the need for further safety assessments of Vanilla cribbiana ethanolic extract. While the extracts of V. bahiana, V. chamissonis, and V. planifolia generally demonstrated non-mutagenic properties in the Ames assay, V. cribbiana exhibited mutagenicity at high concentrations (5000 µg/plate) in the TA98 strain without metabolic activation. This finding, coupled with the dose-dependent cytotoxicity observed in WST-1 (Water Soluble Tetrazolium) assays, a colorimetric method that assesses the viability of cells exposed to a test substance, underscores the importance of concentration in the safety evaluation of these extracts. Kaempferol and pyrogallol, identified with higher intensity in V. cribbiana, are potential candidates for in vitro mutagenicity. Although the results are not conclusive, they suggest the safety of these extracts at low concentrations. This study emphasizes the value of an integrated approach in providing a nuanced understanding of the safety profiles of natural products, advocating for cautious use and further research into V. cribbiana mutagenicity.

Identification of a ß-Carboline Alkaloid from Chemoselectively Derived Vanilla Bean Extract and Its Prevention of Lipid Droplet Accumulation in Human Hepatocytes (HepG2).

Targeting bioactive compounds to prevent lipid droplet accumulation in the liver, we explored an antioxidative extract from vanilla bean (Vainilla planifolia) after chemo-selective derivatization through heating and acid modification. The chemical analysis of vanilla bean extract through chemoselective derivatization resulted in the identification of sixteen compounds (34-50) using LC-MS/MS analysis. A ß-carboline alkaloid with a piperidine C-ring and a vanillin moiety at C-1 (34) was identified by molecular networking and diagnostic fragmentation filtering approaches. ß-carboline alkaloid 34 exhibited significant inhibitory activity of lipid droplet accumulation (LDAI) in oleic acid-loaded hepatocellular carcinoma HepG2 cells. The LDAI activity was associated with both activation of lipolysis and suppression of lipogenesis in the cells. The study indicates that crude plant extracts, following chemoselective derivatization, may contain bioactive compounds that could be beneficial in preventing hepatosteatosis and could serve as a source of lead compounds for drug development. This approach may be useful to investigate other mixtures of natural products and food resources.

Enzymatic Activity and Its Relationships with the Total Phenolic Content and Color Change in the High Hydrostatic Pressure-Assisted Curing of Vanilla Bean (Vanilla planifolia).

Diverse enzymatic reactions taking place after the killing of green vanilla beans are involved in the flavor and color development of the cured beans. The effects of high hydrostatic pressure (HHP) at 50-400 MPa/5 min and blanching as vanilla killing methods were evaluated on the total phenolic content (TPC), polyphenoloxidase (PPO), and peroxidase (POD) activity and the color change at different curing cycles of sweating-drying (C0-C20) of vanilla beans. The rate constants describing the above parameters during the curing cycles were also obtained. The TPC increased from C1 to C6 compared with the untreated green beans after which it started to decrease. The 400 MPa samples showed the highest rate of phenolic increase. Immediately after the killing (C0), the highest increase in PPO activity was observed at 50 MPa (46%), whereas for POD it was at 400 MPa (25%). Both enzymes showed the maximum activity at C1, after which the activity started to decrease. As expected, the L* color parameter decreased during the entire curing for all treatments. An inverse relationship between the rate of TPC decrease and enzymatic activity loss was found, but the relationship with L* was unclear. HHP appears to be an alternative vanilla killing method; nevertheless, more studies are needed to establish its clear advantages over blanching.

A novel magneto-mediated electrochemical biosensor integrated DNAzyme motor and hollow nanobox-like Pt@Ni-Co electrocatalyst as dual signal amplifiers for vanilla detection.

Herein, a novel magneto-mediated electrochemical aptasensor using the signal amplification technologies of DNAzyme motor and electrocatalyst for vanilla (VAN) detection was fabricated. The D/B duplex, formed by the DNAzyme motor that was each silenced by a blocker, and hairpin DNA1 (H1) containing adenosine ribonucleotide (rA) site were tethered on the sites of the gold nanoparticles@hollow porphyrinic-Metal-organic framework/polyethyleneimine-reduced graphene oxide (AuHPCN-222/PEI-rGO)-modified gold electrode (AuE). Then, after homogeneous and specific recognition in the presence of the VAN, trigger DNA was released and enriched by magnetic separation technique and introduced to the sensing platform to activate the DNAzyme motor, which efficiently improved target recognition capability and avoided the obstacle of multiple DNA strands tangling. More interestingly, the activated DNAzyme motor could repeatedly bind to and cleave H1 in the presence of Mg2+, leading to the exposure of a plethora of capture probes. The thionine (Thi) functionalized hairpin DNA2 (H2)-Pt@Ni-Co as signal probes could hybridize with capture probes. Additionally, the Pt@Ni-Co electrocatalysts presented catalytic activity towards Thi to obtain stronger electrochemical signals. VAN with concentrations ranging from 1 × 10-6 to 10 µM was determined and a detection limit was down to 0.15 pM. The designed electrochemical sensor was highly selective with specificity, stability, reproducibility, and reliable capability for monitoring the VAN in real samples.

Sex-Specific Alterations of the Lung Transcriptome at Birth in Mouse Offspring Prenatally Exposed to Vanilla-Flavored E-Cigarette Aerosols and Enhanced Susceptibility to Asthma.

Currently, approximately 8 million adult Americans use electronic cigarettes (e-cigs) daily, including women of childbearing age. It is known that more than 10% of women smoke during their pregnancy, and recent surveys show that rates of maternal vaping are similar to rates of maternal cigarette smoking. However, the effects of inhaling e-cig aerosol on the health of fetuses remain unknown. The objective of the present study was to increase our understanding of the molecular effects caused by in utero exposures to e-cig aerosols on developing mouse lungs and, later in life, on the offspring's susceptibility to developing asthma. METHODS: Pregnant mice were exposed throughout gestation to either filtered air or vanilla-flavored e-cig aerosols containing 18 mg/mL of nicotine. Male and female exposed mouse offspring were sacrificed at birth, and then the lung transcriptome was evaluated. Additionally, once sub-groups of male offspring mice reached 4 weeks of age, they were challenged with house dust mites (HDMs) for 3 weeks to assess asthmatic responses. RESULTS: The lung transcriptomic responses of the mouse offspring at birth showed that in utero vanilla-flavored e-cig aerosol exposure significantly regulated 88 genes in males (62 genes were up-regulated and 26 genes were down-regulated), and 65 genes were significantly regulated in females (17 genes were up-regulated and 48 genes were down-regulated). Gene network analyses revealed that in utero e-cig aerosol exposure affected canonical pathways associated with CD28 signaling in T helper cells, the role of NFAT in the regulation of immune responses, and phospholipase C signaling in males, whereas the dysregulated genes in the female offspring were associated with NRF2-mediated oxidative stress responses. Moreover, we found that in utero exposures to vanilla-flavored e-cig aerosol exacerbated HDM-induced asthma in 7-week-old male mouse offspring compared to respective in utero air + HDM controls. CONCLUSIONS: Overall, these data demonstrate that in utero e-cig aerosol exposure alters the developing mouse lung transcriptome at birth in a sex-specific manner and provide evidence that the inhalation of e-cig aerosols is detrimental to the respiratory health of offspring by increasing the offspring' susceptibility to developing lung diseases later in life.

Carbonyl Profiles of Electronic Nicotine Delivery System (ENDS) Aerosols Reflect Both the Chemical Composition and the Numbers of E-Liquid Ingredients-Focus on the In Vitro Toxicity of Strawberry and Vanilla Flavors.

Propylene glycol (PG) and glycerin (G) are the most widely used humectants in electronic nicotine delivery system (ENDS) devices. Carbonyls are present in aerosols produced when ENDS devices heat PG and G. Whether aerosolized PG and G are innocuous to the lungs has not been established. Here, we determined the chemical profiles of ENDS aerosols containing three humectant ratios (30/70, 50/50 and 70/30, PG/VG), for three flavors (strawberry, vanilla and Catalan cream) containing either 12 or 18 mg/mL of nicotine. Additionally, we examined the in vitro toxicity of the strawberry- and vanilla-flavored ENDS aerosol in human lung epithelial cells (BEAS-2B) exposed at the air-liquid interface for 1 h. For strawberry- and vanilla-flavored aerosols produced by a 3rd-generation ENDS device with the same PG/G ratio, the e-liquid nicotine content of 12 and 18 mg/mL did not transfer to the aerosol with substantial differences in concentrations. Our data also indicate the presence of carbonyls in all three flavored e-cig aerosols analyzed, with levels exceeding 1 µg/puff for acetone, butyraldehyde, and acetaldehyde, in strawberry-, vanilla, and Catalan cream-flavored e-cig aerosols, respectively. Furthermore, closed-system ENDS of the fourth generation emitted trace levels of carbonyls in the aerosols (<0.3 µg/puff), while open-system tank-style ENDS of the third generation produced elevated levels of harmful chemicals, including acrolein (>1 µg/puff), formaldehyde (>5 µg/puff), and m- & p-tolualdehyde (>4 µg/puff). Moreover, under non-cytotoxic conditions, BEAS-2B cells exposed to strawberry-flavored aerosols exhibited significantly increased reactive oxygen and nitric oxide species (ROS/NOS) levels in cell media compared to air controls, while vanilla-flavored ENDS aerosols up-regulated the expression of pro-inflammatory and oxidative stress markers. Our data suggest (a) that ENDS aerosol chemical composition will vary based upon the presence and concentration of the initial e-liquid ingredients, with a pronounced impact of the flavoring components; and (b) short-term exposures to flavored ENDS aerosols may impair lung cells' redox signaling in a flavor-specific manner.

Ohmic heating treatment in high-protein vanilla flavored milk: Quality, processing factors, and biological activity.

The processing of high-protein vanilla-flavored milk was performed under different electric field strengths of ohmic heating (5.22 V/cm, OH6; 6.96 V/cm, OH8; 8.70 V/cm, OH10; 10.43 V/cm, OH12) to evaluate the energy consumption, processing parameters, and microbiological, rheological, and biological aspects, compared with the sample submitted to conventional pasteurization (PAST, 72 °C/15 s). All samples showed higher than 12 g/100 mL of protein, consisting of high-protein content products. In addition, Ohmic Heating (OH) generated lower energy expenditure and more significant microbial inactivation of lactic acid bacteria, molds and yeasts, total mesophiles, and psychotropics. Furthermore, OH at lower electric field strengths, mainly OH8, improved anti-diabetic, anti-oxidant, and anti-hypertensive activities and rheological properties, and resulted in lower hydroxymethylfurfural contents, and higher whey protein nitrogen index. The results suggest that OH is a technology that can be used in flavored milk with high-protein content, being recommended an electric field strength of 6.96 V/cm. However, more studies are necessary to evaluate the effect of OH on high-protein dairy products, mainly by studying other OH processing parameters.

Comparison of the analgesic effect of inhaled lavender vs vanilla essential oil for neonatal frenotomy: a randomized clinical trial (NCT04867824).

It is necessary to treat neonatal pain because it may have short- and long-term adverse effects. Frenotomy is a painful procedure where sucking, a common strategy to relieve pain, cannot be used because the technique is performed on the tongue. In a previous randomized clinical trial, we demonstrated that inhaled lavender essential oil (LEO) reduced the signs of pain during neonatal frenotomy. We aimed to find out whether inhaled vanilla essential oil (VEO) is more effective in reducing pain during frenotomy than LEO. Randomized clinical trial with neonates who underwent a frenotomy for type 3 tongue-ties between May and October 2021. Pain was assessed using pre and post-procedure heart rate (HR) and oxygen saturation (SatO2), crying time, and NIPS score. Neonates were randomized into "experimental" and "control" group. In both groups, we performed swaddling, administered oral sucrose, and let the newborn suck for 2 min. We placed a gauze pad with one drop of LEO (control group) or of VEO (experimental group) under the neonate's nose for 2 min prior to and during the frenotomy. We enrolled 142 neonates (71 per group). Both groups showed similar NIPS scores (2.02 vs 2.38) and crying times (15.3 vs 18.7 s). We observed no differences in HR increase or in SatO2 decrease between both groups. We observed no side effects in either of the groups. CONCLUSIONS: We observed no appreciable difference between LEO and VEO; therefore, we cannot conclude which of them was more effective in treating pain in neonates who underwent a frenotomy. TRIAL REGISTRATION:  This clinical trial is registered with www. CLINICALTRIALS: gov with NCT04867824. WHAT IS KNOWN: • Pain management is one of the most important goals of neonatal care as it can have long-term neurodevelopmental effects. • Lavender essential oil can help relieve pain due to its sedative, antispasmodic, and anticolic properties. WHAT IS NEW: • Lavender and vanilla essential oils are safe, beneficial, easy to use, and cheap in relieving pain in neonates who undergo a frenotomy for type 3 tongue-ties.

In vitro response of vanilla (Vanilla planifolia Jacks. ex Andrews) to PEG-induced osmotic stress.

Drought-induced water stress affects the productivity of the Vanilla planifolia Jacks. ex Andrews crop. In vitro culture technique is an effective tool for the study of water stress tolerance mechanisms. This study aimed to evaluate the morphological, physiological and biochemical response of V. planifolia under in vitro water stress conditions induced with polyethylene glycol (PEG). In vitro regenerated shoots of 2 cm in length were subjected to different concentrations of PEG 6000 (0, 1, 2 and 3% w/v) using Murashige and Skoog semi-solid culture medium. At 60 days of culture, different growth variables, dry matter (DM) content, chlorophyll (Chl), soluble proteins (SP), proline (Pro), glycine betaine (GB), stomatal index (SI) and open stomata (%) were evaluated. Results showed a reduction in growth, Chl content, SP, SI and open stomata (%) with increasing PEG concentration, whereas DM, Pro and GB contents rose with increasing PEG concentration. In conclusion, PEG-induced osmotic stress allowed describing physiological and biochemical mechanisms of response to water stress. Furthermore, the determination of compatible Pro and GB osmolytes can be used as biochemical markers in future breeding programs for the early selection of water stress tolerant genotypes.

Vanilla scent reduces frequency of urination in urethane-anesthetized rats.

OBJECTIVES: The scent of vanilla has a relaxing effect and is used to treat sleep disorders. Sleep disorders can both cause and be caused by nocturia. Therefore, we examined whether vanilla inhalation would reduce the frequency of urination in rats under light urethane anesthesia. METHODS: Twenty-four rats were anesthetized with 0.6 g/kg urethane subcutaneously (half the usual dose) to induce a sleep-like state. In 12 rats, continuous cystometry was performed via a transurethral catheter before, during and after inhalation of vanilla (n = 7) or the citrus fruit shiikuwasa (n = 5) for 60 minutes. The remaining 12 rats did not undergo cystometry but underwent vanilla inhalation treatment for 60 minutes (n = 6), or no inhalation treatment (n = 6); blood was then collected from these two groups and serum monoamine levels were compared. RESULTS: Intervals between bladder contractions were significantly longer after vanilla inhalation than before. However, baseline bladder pressure, maximum bladder contraction pressure, and residual volume remained unchanged. During shiikuwasa inhalation, the body movement of each rat increased but cystometric parameters did not change. Serum concentrations of adrenaline, noradrenaline and dopamine, but not serotonin, were significantly lower in rats that had inhaled vanilla than in those that had not. CONCLUSIONS: Vanilla scent decreased serum catecholamine levels and urination frequency in rats under light urethane anesthesia. These results suggest that the scent of vanilla may reduce nocturia.

First record of Vanilla labellopapillata (Orchidaceae: Vanilloideae) in the state of Amazonas, Brazil

The first record of Vanilla labellopapillata is presented for the state of Amazonas, Brazil, in the region of Manaus, now the western limit of the species, which was previously known only from the type locality in the state of Pará. A brief description is provided and taxonomic and ecological aspects of the species are discussed in the light of this new finding. (AU)

Permeability of the amniotic membranes to the natural vanilla molecule.

Purpose: We studied natural vanilla permeability through amniotic membranes.Methods: We studied natural vanilla permeability through amniotic membranes obtained from 45 spontaneous normal deliveries at term. The ferric chloride test (FeCL3) was used to determine the presence or absence of phenols in a given sample. Vanilla is a polyphenol so it gives a reaction to FeCL3 with an intense color change. The diffusion of the vanilla was checked by dropping ferric chloride solution on the gauze once the membranes are lifted with care to avoid contamination. If vanilla has passed through the membranes the distilled water papers would change from an initial ferric yellow in the drops toward a marked gray/greenish color on the papers (positive test).Results: In all cases, the swabs were stained, all the membranes in both directions were permeable to the whole vanilla molecule.Conclusions: This experiment allows us to reevaluate the importance of molecular diffusion through the amniotic membranes with no placental metabolism existing between maternal and fetal environment.

Efecto de la interacción luz-agua sobre la fotosíntesis de la Vanilla planifolia (Orchidaceae) / Light-water interaction effect on photosynthesis of the Vanilla planifolia (Orchidaceae)

Introducción: La vainilla (Vanilla planifolia Andrews) es una planta CAM de gran importancia económica en el mercado mundial de saborizantes y aromatizantes. Por ser una planta hemiepífita que inicialmente crece en el sotobosque sombreado y posteriormente asciende hasta el dosel, se aclimata a diferentes condiciones de radiación y humedad. La posibilidad de extender su cultivo a zonas secas donde sea menor la incidencia de enfermedades, dependerá de su respuesta a períodos prolongados de sequía. Objetivo: Evaluar los efectos de la interacción entre la radiación y la humedad sobre los parámetros de la fotosíntesis y funcionamiento del CAM de plantas de vainilla en sus fases iniciales de desarrollo vegetativo, y determinar si las condiciones de radiación en las que crece la planta pueden ayudar a moderar los efectos negativos de la sequía. Métodos: Se evaluaron plantas de vainilla que crecieron con riego durante 18 meses en casetas con iluminación relativa (IR) de 8, 17, 31 y 67 %; posteriormente se suspendió el riego y se fueron deshidratando durante 94 días hasta alcanzar un contenido de humedad crítico (contenido hídrico relativo, CHR ≤ 50 %), y finalmente se reinició el riego durante 22 días; se evaluó el efecto de estos procesos sobre la asimilación de CO2, la acidez titulable y la eficiencia cuántica del Fotosistema II (Fv/Fm). Resultados: Bajo condiciones intermedias de IR (17 % y 31 %), la asimilación de CO2 y laacumulación nocturna de ácidos orgánicos presentaron la menor afectación por el déficit hídrico, así como la mejor recuperación luego de la rehidratación. El CHR se vio más afectado por la disponibilidad de agua en el tejido vegetal, mientras que Fv/Fm lo fue por el ambiente de radiación. Los efectos combinados de estrés hídrico y alta radiación ocasionaron daños irreversibles en la fotosíntesis para el tratamiento de IR de 67 %. Conclusiones: En las plantas de vainilla, el impacto negativo de la sequía sobre los parámetros de la fotosíntesis fue mayor en alta radiación; sin embargo, en condiciones de baja radiación también aumentó la susceptibilidad de las plantas a la sequía, en comparación con los ambientes de radiación intermedia, en los cuales la densidad de flujo de fotones media fue de 340 μmol m-2 seg-1 (17 % de IR) y 620 μmol m-2 seg-1 (31 % de IR). Estos resultados sugieren el potencial de cultivar vainilla en zonas con sequía estacional bajo sistemas productivos de baja tecnificación, manteniendo estas condiciones de radiación.

Introduction: Vanilla planifolia Andrews is a CAM plant of economic importance in the global market of flavorings and essences. Being a hemiepiphyte which initially grows in the shady understory and later climbs to the canopy, it acclimates to different conditions of radiation and humidity. The possibility of extending this crop to dry areas where the incidence of diseases is lower will depend on the response to prolonged periods of drought. Objective: To evaluate the effects of the interaction of radiation and humidity on the parameters of photosynthesis and operation of CAM of vanilla plants, and to determine if the radiation environment could help to moderate the negative effects of drought. Methods: We evaluated well-watered vanilla plants grown for 18 months in relative illumination (RI) of 8, 17, 31 and 67 %, which underwent dehydration for 94 days until reaching critical water content (relative water content, RWC < 50 %) and then rehydration for 22 days; variables evaluated were: CO2 assimilation, titratable acidity, and quantum efficiency of Photosystem II (Fv / Fm). Results: Under intermediate conditions of radiation (17 % and 31 % RI), CO2 assimilation and nocturnal accumulation of organic acids were less affected by water deficit and also showed the best recovery after rehydration. The RWC was most affected by the water available in plant tissue, while Fv / Fm was affected by radiation. The combined effects of water stress and high radiation caused irreversible damage to photosynthesis for the 67 % RI treatment. Conclusions: In vanilla plants, the negative impact of drought on photosynthetic parameters was greater in high radiation; however, in low radiation conditions the susceptibility of plants to drought also increased, as compared to intermediate radiation environments, which were under a mean photon flux density of 340 µmol m-2 s-1 (17 % IR) and 620 µmol m-2 s-1 (31 % IR). These results suggest the potential for growing vanilla in areas with seasonal drought under low-tech production systems, maintaining these radiation conditions.

Metabolite Transformation and Enzyme Activities of Hainan Vanilla Beans During Curing to Improve Flavor Formation.

This paper compares the differences in metabolites of vanilla beans at five different curing stages. Key vanilla flavors, vanillin precursors and main enzymes during the curing process of Hainan vanilla beans were also analyzed. Hundreds of metabolites were detected based on metabolic analyses of a widely targeted metabolome technique, compared with blanched vanilla beans (BVB), sweating vanilla beans (SVB) and drying vanilla beans (DVB), the total peak intensity of cured vanilla beans (CVB) is on the rise. The score plots of principal component analysis indicated that the metabolites were generally similar at the same curing stages, but for the different curing stages, they varied substantially. During processing, vanillin content increased while glucovanillin content decreased, and vanillic acid was present in sweating beans, but its content was reduced in drying beans. Both p-hydroxybenzaldehyde and p-hydroxybenzoic acid showed the maximum contents in cured beans. Ferulic acid was mainly produced in drying beans and reduced in cured beans. p-coumaric acid increased during the curing process. Vanillyl alcohol in drying beans (0.22%) may be formed by the hydrolysis of glucoside, whose conversion into vanillin may explain its decrease during the curing stage. ß-Glucosidase enzymatic activity was not detected in blanched and sweating beans, but was observed after drying. Peroxidase activity decreased during curing by 94% in cured beans. Polyphenol oxidase activity was low in earlier stages, whereas cellulase activity in processed beans was higher than in green beans, except for cured beans. This study contributes to revealing the formation of flavor components and the biosynthesis pathway of vanillin.

The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia.

Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia.

Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH4)2SO4 precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe2+. Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia.

A re-evaluation of the final step of vanillin biosynthesis in the orchid Vanilla planifolia.

A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a "vanillin synthase" could enable biotechnological production of vanillin from ferulic acid using a "natural" vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined.