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.

Impact of the Post-Harvest Period on the Chemical and Sensorial Properties of planifolia and pompona Vanillas.

Vanilla production in Guadeloupe is expanding. The main species grown is Vanilla planifolia, but other species such as Vanilla pompona are also present and required by industries. To upgrade the value of vanilla production on this Caribbean Island, this study was performed to evaluate the aromatic specifies of these vanilla species according to the length of the post-harvest period (2 months and 9 months). For this purpose, Vanilla planifolia and Vanilla pompona were compared through scald and scarification transformation processes, as well as two different refining times (T1 and T2). For chemical characterization, 0.1 g of vanilla bean seeds was used for SMPE/GC-MS measurements, while 0.05 g of vanilla samples was subjected to infusion in milk (0.15%) for sensory evaluation. The latter involved generation of terms of aroma through olfaction and gustation sessions. The chemical results showed a significant difference between the two species, where vanillin was mostly present in Vanilla planifolia, unlike Vanilla pompona, where it was mainly rich in 4-methoxybenzyl alcohol. Interestingly, the second refining time was characterized by the appearance of two major components, 1,3-octadien and acetic acid. For sensory analysis, all the vanillas exhibited a high diversity of aromas including "sweet", "gourmand", "spicy" flavors and so on. The application of factorial correspondence analysis (FAC) as well as the agglomerative hierarchical clustering (AHC) showed differences between the vanilla samples according to both the species and refining time. The combination of these analyses makes it possible to establish a chemical and organoleptic profile of vanillas. Varietal and processing factors both have a major impact on the aroma profile of vanillas.

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.

Recovery of flavor compounds from vanilla bagasse by hydrolysis and their identification through UPLC-MSE.

Vanilla is a globally treasured commodity, and the consequences of its unstable value affect social, environmental, economic, and academic ambits. The extensive range of aroma molecules found in cured vanilla beans is crucial to the complexity of this natural condiment and knowledge about their recovery is of the essence. Many strategies aim on reproducing the chemical intricacies of vanilla flavor, such as biotransformation and de novo biosynthesis. Few studies, however, aim at the exhaustion of the cured pods, of which the bagasse, after the traditional ethanolic extraction, might still bear a highly valued flavor composition. An untargeted liquid chromatography coupled with mass spectrometry (LC-MSE) approach was applied to elucidate if sequential alkaline-acidic hydrolysis was effective in extracting flavor related molecules and chemical classes from the hydro-ethanolic fraction. Important vanilla flavor related compounds present in the hydro-ethanolic fraction were further extracted from the residue through alkaline hydrolysis, such as vanillin, vanillic acid, 3-methoxybenzaldehyde, 4-vinylphenol, heptanoic acid, and protocatechuic acid. Acid hydrolysis was effective on further extracting features from classes such as phenols, prenol lipids, and organooxygen compounds, though representative molecules remain unknown. Finally, sequential alkaline-acidic hydrolysis rendered natural vanilla's ethanolic extraction residues as an interesting supplier of its own products, which could be used as a food additive, and many other applications.

Quantitative characterizations of mOR-EG activated by vanilla odorants using advanced statistical physics modeling.

An advanced monolayer adsorption model of an ideal gas was successfully employed to investigate the adsorption of vanillin, vanillin methyl ether, vanillin ethyl ether, and vanillin acetate odorants on mouse eugenol olfactory receptor mOR-EG. In order to understand the adsorption process putatively introduced in olfactory perception, model parameters were analyzed. Hence, fitting results showed that the studied vanilla odorants were linked in mOR-EG binding pockets with a non-parallel orientation, and their adsorption was a multi-molecular process (n > 1). The adsorption energy values that ranged from 14.021 to 19.193 kJ/mol suggested that the four vanilla odorants were physisorbed on mOR-EG (ΔEa < 40 kJ/mol) and the adsorption mechanism may be considered as an exothermic mechanism (ΔEa > 0). The estimated parameters may also be utilized for the quantitative characterization of the interactions of the studied odorants with mOR-EG to determine the corresponding olfactory bands ranging from 8 to 24.5 kJ/mol.

Rapid Sample Screening Method for Authenticity Controlling of Vanilla Flavours Using Liquid Chromatography with Electrochemical Detection Using Aluminium-Doped Zirconia Nanoparticles-Modified Electrode.

A rapid and sensitive technique for frauds determination in vanilla flavors was developed. The method comprises separation by liquid chromatography followed by an electrochemical detection using a homemade screen-printed carbon electrode modified with aluminium-doped zirconia nanoparticles (Al-ZrO2-NPs/SPCE). The prepared nanomaterials (Al-ZrO2-NPs) were characterized by using X-ray diffraction (XRD), transmission electron microscopy (TEM) and energy dispersive X-ray (EDX). This method allows for the determination of six phenolic compounds of vanilla flavors, namely, vanillin, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillyl alcohol, vanillic acid and ethyl vanillin in a linear range between 0.5 and 25 µg g-1, with relative standard deviation values from 2.89 to 4.76%. Meanwhile, the limits of detection and quantification were in the range of 0.10 to 0.14 µg g-1 and 0.33 to 0.48 µg g-1, respectively. In addition, the Al-ZrO2-NPs/SPCE method displayed a good reproducibility, high sensitivity, and good selectivity towards the determination of the vanilla phenolic compounds, making it suitable for the determination of vanilla phenolic compounds in vanilla real extracts products.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due to the availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes were confirmed through a ß-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) µg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Effect-directed profiling of 32 vanilla products, characterization of multi-potent compounds and quantification of vanillin and ethylvanillin.

Food testing is of great importance to the food industry and organizations to verify the authenticity claims, to prove the quality of raw materials and products, and to ensure food safety. The market prices of vanilla differed by a factor of about 20 in the last three decades. Therefore the risk of adulteration and counterfeiting of vanilla products is high. Instead of commonly used target analyses and sum parameter assays, a complementary non-target multi-imaging effect-directed screening was developed, which provided a new perspective on the wide range of vanilla product qualities on the market. Planar chromatography was combined with effect-directed assays, and the obtained biological and biochemical profiles of 32 vanilla products from nine different categories revealed a variety of active ingredients. Depending on the region, typical vanilla product profiles and activity patterns were obtained for pods, tinctures, paste (inner part), oleoresin and powders. However, some vanilla products showed additional active compounds and a different intensity pattern. The vanilla product profiles substantially differed from those of vanilla aroma or products containing synthetic vanillin or vanilla-flavored food products. Bioactive compounds of interest were online eluted and further characterized via HPTLC-HRMS, which allowed their tentative assignment. After purchase of the standards, these were successfully confirmed by co-chromatography. Quantification of vanillin across nine different product categories revealed levels ranging from 1 µg/g to 36 mg/g with a mean repeatability of 1.9%. The synthetic ethylvanillin was not detected in the investigated samples in significant concentrations. The assessment of differences in the activity patterns pointed to highly active compounds, which were not detected at UV/Vis/FLD but first via the biological and enzymatic assays. This effect-directed profiling bridges the gap from analytical food chemistry to food toxicology, and thus, makes an important contribution to consumer safety. In the same way, it would accelerate investigations for Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) according to Regulation (EC) No. 1907/2006.

New Insights on Volatile Components of Vanilla planifolia Cultivated in Taiwan.

Vanilla (Vanilla planifolia) is a precious natural flavoring that is commonly used throughout the world. In the past, all vanilla used in Taiwan was imported; however, recent breakthroughs in cultivation and processing technology have allowed Taiwan to produce its own supply of vanilla. In this study, headspace solid-phase microextraction (HS-SPME) combined with GC-FID and GC-MS was used to analyze the volatile components of vanilla from different origins produced in Taiwan under different cultivation and processing conditions. The results of our study revealed that when comparing different harvest maturities, the composition diversity and total volatile content were both higher when the pods were matured for more than 38 weeks. When comparing different killing conditions, we observed that the highest vanillin percentage was present after vanilla pods were killed three times in 65 °C treatments for 1 min each. From the experiment examining the addition of different strains, the PCA results revealed that the volatiles of vanilla that was processed with Dekkera bruxellensis and Bacillus subtilis was clearly distinguished from which obtained by processing with the other strains. Vanilla processed with B. subtilis contained 2-ethyl-1-hexanol, and this was not detected in other vanillas. Finally, when comparing the vanillin percentage from seven different regions in Taiwan, vanilla percentage from Taitung and Taoyuan Longtan were the highest.

NMR-based leaf metabolic profiling of V. planifolia and three endemic Vanilla species from the Peruvian Amazon.

The fruit of Vanilla planifolia is broadly preferred by the agroindustry and gourmet markets due to its refined flavor and aroma. Peruvian Vanilla has been proposed as a possible source for genetic improvement of existing Vanilla cultivars, but, little has been done to facilitate comprehensive studies of these and other Vanilla. Here, a nuclear magnetic resonance (NMR) metabolomic platform was developed to profile for the first time the leaves - organ known to accumulate vanillin putative precursors - of V. planifolia and those of Peruvian V. pompona, V. palmarum, and V. ribeiroi, with the aim to determine metabolic differences among them. Analysis of the NMR spectra allowed the identification of thirty-six metabolites, twenty-five of which were quantified. One-way ANOVA and post-hoc Tukey test revealed that these metabolites changed significantly among species, whilst multivariate-analyses allowed the identification of malic and homocitric acids, together with two vanillin precursors, as relevant metabolic markers for species differentiation.

Discrimination of sensory attributes by trained assessors and consumers in semi-sweet hard dough biscuits and their drivers of liking and disliking.

Descriptive methods are traditionally performed with trained assessors to objectively analyze products, since consumers have long been considered incapable of performing such tests because they are influenced by hedonic evaluations. However, in the last decades, there have been alternative descriptive tests performed by consumers, due to other advantages, such as a rapid sensory profile assessment, reducing cost and time, and Check-all-that-apply (CATA) is an example. Thus, the objective of this work was to compare the performance of trained assessors (n = 15) and consumers using quantitative descriptive analysis (QDA) - traditional method and CATA - alternative method (n = 161 consumers, including acceptance test as well), for 5 samples of semi-sweet hard dough biscuit with different contents of specific ingredients: fructose (0.0 to 6.0%), vanilla aroma (0.0 to 1.2%) and water (9.0 to 13.8%). Results showed that the number of attributes in which trained assessors perceived significant differences was marginally higher (p < .1) than consumers. For smaller formulation differences, trained assessors perceived more (p < .01) attributes presenting significant differences (10) than consumers (2). The sample discrimination presented the same pattern, however trained assessors had greater discriminative power than consumers, i.e., they discriminated samples in more difference levels. The description of the samples was not similar between the methods. The QDA and CATA assessors used different attributes to describe the samples, which reflected in different drivers of liking and disliking if descriptive data come from trained assessors or from consumers. It is concluded that the choice of performing descriptive tests with trained assessors or consumers depends on the purpose of the study.

Vanilla bahiana, a contribution from the Atlantic Forest biodiversity for the production of vanilla: A proteomic approach through high-definition nanoLC/MS.

Only a few cultivated species of Vanilla are used to produce vanilla, despite the high demand, predatory exploitation, and low genetic variability that threaten the production of natural vanilla. Vanilla bahiana pods from the Atlantic Forest may be an alternative source of natural vanilla. This study applied bottom-up and shotgun proteomics analysis to identify proteins related to flowering, fruiting, and vanilla-flavor production. Extraction solutions, including Tris-HCl buffer, ß-mercaptoethanol and SDS, were assayed. SDS proved to be feasible for extraction of Vanilla fruit proteins and could be an alternative to the phenol method of protein extraction. Progenesis QI for Proteomics (QIP) software loaded with an Orchidaceae database identified 2326 proteins in our samples. Among these, 75 were highlighted as useful for the synthesis of compounds related to vanilla flavor, such as vanillin synthase, which was successfully extracted with 1% SDS, which also improved the variety of the extracted proteins. The proteins identified in V. bahiana pods indicate the enzymatic potential of this species, as further validated by quantifying the vanilla in the samples.

Capillary electrophoresis method for the discrimination between natural and artificial vanilla flavor for controlling food frauds.

A capillary electrophoresis method was developed for the determination of coumarin (COUM), ethyl vanillin (EVA), p-hydroxybenzaldehyde (PHB), p-hydroxybenzoic acid (PHBA), vanillin (VAN), vanillic acid (VANA) and vanillic alcohol (VOH) in vanilla products. The measured concentrations are compared to values obtained by liquid chromatography (LC) method. Analytical results, method precision, and accuracy data are presented and limits of detection for the method ranged from 2 to 5 µg/mL. The results obtained are used in monitoring the composition of vanilla flavorings, as well as for confirmation of natural or non-natural origin of vanilla in samples using four selected food samples containing this flavor.

Sweet Emotion: The Role of Odor-induced Context in the Search Advantage for Happy Facial Expressions.

The current study investigated the extent to which the concurrent presentation of pleasant and unpleasant odors could modulate the perceptual saliency of happy facial expressions in an emotional visual search task. Whilst a search advantage for happy faces was found in the no odor and unpleasant odor conditions, it was abolished under the pleasant odor condition. Furthermore, phasic properties of visual search performance revealed the malleable nature of this happiness advantage. Specifically, attention towards happy faces was optimized at the start of the visual search task for participants presented with pleasant odors, but diminished towards the end. This pattern was reversed for participants in the unpleasant odor condition. These patterns occur through the emotion-inducing capacities of odors and highlight the circumstances in which top-down factors can override perceptually salient facial features in emotional visual search.

Determination of major aromatic constituents in vanilla using an on-line supercritical fluid extraction coupled with supercritical fluid chromatography.

An on-line supercritical fluid extraction coupled with supercritical fluid chromatography method was developed for the determination of four major aromatic constituents in vanilla. The parameters of supercritical fluid extraction were systematically investigated using single factor optimization experiments and response surface methodology by a Box-Behnken design. The modifier ratio, split ratio, and the extraction temperature and pressure were the major parameters which have significant effects on the extraction. While the static extraction time, dynamic extraction time, and recycle time had little influence on the compounds with low polarity. Under the optimized conditions, the relative extraction efficiencies of all the constituents reached 89.0-95.1%. The limits of quantification were in the range of 1.123-4.747 µg. The limits of detection were in the range of 0.3368-1.424 µg. The recoveries of the four analytes were in the range of 76.1-88.9%. The relative standard deviations of intra- and interday precision ranged from 4.2 to 7.6%. Compared with other off-line methods, the present method obtained higher extraction yields for all four aromatic constituents. Finally, this method has been applied to the analysis of vanilla from different sources. On the basis of the results, the on-line supercritical fluid extraction-supercritical fluid chromatography method shows great promise in the analysis of aromatic constituents in natural products.

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.

Novel Creation of a Rum Flavor Lexicon Through the Use of Web-Based Material.

Flavor lexicons help both manufacturers and consumers communicate the intricacies of flavor nuances they experience within a product. Lexicon development typically requires the use of a trained sensory panel to evaluate a representative sample set of the product category to generate terms that describe certain product attributes. In the case of rum, there is considerable variation in terms of style, flavor characteristics, and the sheer number of rums produced making it difficult to create a lexicon in this manner. Furthermore, sensory fatigue from the high alcohol content can also hinder lexicon development. This is the first study to create a rum flavor lexicon using web-based material (comprising blogs, company descriptions, and review websites) to minimize the time and cost and to allow for the inclusion of a greater number of rum products. Reviews for over 1000 different rums were utilized, comprising evaluations that described an array of rums, including white, gold, aged, and agricole. Each evaluation was coded for aroma, aroma-by-mouth, and taste attributes using NVivoTM software to amass the sensory terms. Word frequency analysis was conducted on coded attributes. The analysis yielded 147 terms, sorted into 22 different categories. The most prominent terms included vanilla, oak, caramel, fruity, molasses, and baking spices.

Suppression of cancer stem-like phenotypes in NCI-H460 lung cancer cells by vanillin through an Akt-dependent pathway.

Cancer stem cells (CSCs) have been reported as a major cause of cancer metastasis and the failure of cancer treatment. Cumulative studies have indicated that protein kinase B (Akt) and its downstream signaling pathway, including CSC markers, play a critical role in the aggressive behavior of this cancer. In this study, we investigated whether vanillin, a major component in Vanilla planifolia seed, could suppress cancer stemness phenotypes and related proteins in the human non-small cell lung cancer NCI­H460 cell line. A non-toxic concentration of vanillin suppressed spheroid and colony formation, two hallmarks of the cancer stemness phenotype, in vitro in NCI­H460 cells. Western blot analysis revealed that the CSC markers CD133 and ALDH1A1 and the associated transcription factors, Oct4 and Nanog, were extensively downregulated by vanillin. Vanillin also attenuated the expression and activity of Akt, a transcription regulator upstream of CSCs, an action that was confirmed by treatment with the Akt inhibitor perifosine. Furthermore, the ubiquitination of Akt was elevated in response to vanillin treatment prior to proteasomal degradation. This finding indicates that vanillin can inhibit cancer stem cell-like behavior in NCI­H460 cells through the induction of Akt-proteasomal degradation and reduction of downstream CSC transcription factors. This inhibitory effect of vanillin may be an alternative approach in the treatment against lung cancer metastasis and its resistance to chemotherapy.

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin.

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately 200 different odorant compounds in addition to vanillin. The natural extraction of vanillin (from the orchid Vanilla planifolia, Vanilla tahitiensis and Vanilla pompon) represents only 1% of the worldwide production and since this process is expensive and very long, the rest of the production of vanillin is synthesized. Many biotechnological approaches can be used for the synthesis of vanillin from lignin, phenolic stilbenes, isoeugenol, eugenol, guaicol, etc., with the disadvantage of harming the environment since these processes use strong oxidizing agents and toxic solvents. Thus, eco-friendly alternatives on the production of vanillin are very desirable and thus, under current investigation. Porous coordination polymers (PCPs) are a new class of highly crystalline materials that recently have been used for catalysis. HKUST-1 (Cu3(BTC)2(H2O)3, BTC = 1,3,5-benzene-tricarboxylate) is a very well known PCP which has been extensively studied as a heterogeneous catalyst. Here, we report a synthetic strategy for the production of vanillin by the oxidation of trans-ferulic acid using HKUST-1 as a catalyst.