Chasing the chill: Unveiling the dynamic flavors and textures of ice cream formulations.

In this study, nine commercial ice creams and four prototypes were assessed. For the sensory analysis, quantitative descriptive analysis (QDA), and temporal dominance of sensations (TDS) methodology were used. According to the QDA results, full-composition ice cream showed significantly higher differences (p < .05) in terms such as vanilla and sweet flavors, brightness, creamy texture and appearance, and viscosity, exhibiting longer melting times. Functional ice creams showed significantly higher differences (p < .05) in terms like viscosity, creamy texture, and appearance. On the other hand, agave fructans in prototype ice creams were found to be able to reduce fat, but not fat and sugar simultaneously, showing a significant decrease (p < .05) in terms such as hardness, crystallized texture, gummy texture, and porosity. Based on the PCA results, the analysis accounted for 75.28% of data variability. Full-composition ice creams and one functional ice cream were related to terms such as viscosity, fatty sensation, creamy texture and appearance, dense, gummy, among others. The rest of the commercial ice creams were related to vanilla and caramel flavor and smell, artificial aftertaste, aerated, porosity, among others. Prototype ice creams were related to hard texture, salty and milk flavor, and crystallized texture. Based on the results of the TDS method, all formulations were found to be significantly dominant (5%) in terms such as vanilla flavor and sweet flavor at the beginning of the test. Formulations containing butyric fat and/or vegetable fat, or agave fructans were significantly dominant (5%) in fatty sensation.

Frequency and behavior of Melipona stingless bees and orchid bees (Hymenoptera: Apidae) in relation to floral characteristics of vanilla in the Yucatán region of Mexico.

Vanilla planifolia is native to the Mexican tropics. Despite its worldwide economic importance as a source of vanilla for flavoring and other uses, almost all vanilla is produced by expensive hand-pollination, and minimal documentation exists for its natural pollination and floral visitors. There is a claim that vanilla is pollinated by Melipona stingless bees, but vanilla is more likely pollinated by orchid bees. Natural pollination has not been tested in the Yucatán region of Mexico, where both vanilla and potential native bee pollinators are endemic. We document for the first time the flowering process, nectar production and natural pollination of V. planiflora, using bagged flower experiments in a commercial planting. We also assessed the frequency and visitation rates of stingless bees and orchid bees on flowers. Our results showed low natural pollination rates of V. planifolia (~ 5%). Only small stingless bees (Trigona fulviventris and Nannotrigona perilampoides) were seen on flowers, but no legitimate visits were witnessed. We verified that there were abundant Euglossa and fewer Eulaema male orchid bees around the vanilla plants, but neither visited the flowers. The introduction of a colony of the stingless bee Melipona beecheii and the application of chemical lures to attract orchid bees failed to induce floral visitations. Melipona beecheii, and male orchid bees of Euglossa viridissima and E. dilemma may not be natural pollinators of vanilla, due to lack of attraction to flowers. It seems that the lack of nectar in V. planifolia flowers reduces the spectrum of potential pollinators. In addition, there may be a mismatch between the attractiveness of vanilla floral fragrances to the species of orchid bees registered in the studied area. Chemical studies with controlled experiments in different regions would be important to further elucidate the potential pollinators of vanilla in southern Mexico.

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.

Distinct orchid mycorrhizal fungal communities among co-occurring Vanilla species in Costa Rica: root substrate and population-based segregation.

Despite being the second largest family of flowering plants, orchids represent community structure variation in plant-microbial associations, contributes to niche partitioning in metacommunity assemblages. Yet, mycorrhizal communities and interactions remain unknown for orchids that are highly specialized or even obligated in their associations with their mycorrhizal partners. In this study, we sought to compare orchid mycorrhizal fungal (OMF) communities of three co-occurring hemiepiphytic Vanilla species (V. hartii, V. pompona, and V. trigonocarpa) in tropical forests of Costa Rica by addressing the identity of their OMF communities across species, root types, and populations, using high-throughput sequencing. Sequencing the nuclear ribosomal internal transcribed spacer (nrITS) yielded 299 fungal Operational Taxonomic Units (OTUs) from 193 root samples. We showed distinct segregation in the putative OMF (pOMF) communities of the three coexisting Vanilla hosts. We also found that mycorrhizal communities associated with the rare V. hartii varied among populations. Furthermore, we identified Tulasnellaceae and Ceratobasidiaceae as dominant pOMF families in terrestrial roots of the three Vanilla species. In contrast, the epiphytic roots were mainly dominated by OTUs belonging to the Atractiellales and Serendipitaceae. Furthermore, the pOMF communities differed significantly across populations of the widespread V. trigonocarpa and showed patterns of distance decay in similarity. This is the first report of different pOMF communities detected in roots of wild co-occurring Vanilla species using high-throughput sequencing, which provides evidence that three coexisting Vanilla species and their root types exhibited pOMF niche partitioning, and that the rare and widespread Vanilla hosts displayed diverse mycorrhizal preferences.

Large-Scale Micropropagation of Vanilla (Vanilla planifolia Jacks.) in a Temporary Immersion Bioreactor (TIB).

The cultivation of vanilla (Vanilla planifolia) is of economic interest because vanillin is extracted from the fruits of this species. Vanillin is a natural flavoring highly valued in the food market. However, there is a short supply of propagules available for establishing commercial plantations and good-quality plants with phytosanitary certification. Plant tissue culture represents a viable option to supply large amounts of healthy plants to vanilla producers. In addition, the use of temporary immersion systems will allow commercial scale-up and the establishment of biofactories dedicated to in vitro vanilla propagation. This chapter describes a large-scale micropropagation protocol for vanilla using temporary immersion bioreactors (TIB).

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.

Non-species-specific pollen transfer and double-reward production in euglossine-pollinated Vanilla.

Commonly attributed to orchids, the pollen movement in Vanilla has been associated with food deception and specific plant-pollinator relationships. This study investigated the role of flower rewards and pollinator specificity in the pollen transfer of a widely distributed member to the euglossinophilous Vanilla clade, V. pompona Schiede using data collected from Brazilian populations. These included investigations on morphology, light microscopy and histochemistry, and analysis of flowers scent using GC-MS. The pollinators and the mechanisms of pollination were recorded through focal observations. The yellow flowers of V. pompona are fragrant and offer nectar as reward. The major volatile compound of the V. pompona scent, carvone oxide, shows convergent evolution in Eulaema-pollinated Angiosperms. The pollination system of V. pompona is not species-specific, but its flowers are strongly adapted to pollination by large Eulaema males. Pollination mechanism is based in a combination of perfume collection and nectar seeking. The dogma of a species-specific pollination system based on food deception in Vanilla has been broken with the increase in studies on this Pantropical orchid genus. Here, least three bee species and dual reward-offering are involved in pollen transfer in V. pompona. Visitation frequency of bees collecting perfumes, used in courtship by male euglossines, is higher than in searching for food, as short-lived young euglossine males seem to be more interested in sex than food. A pollination system based on offering both nectar and perfumes as resources is described for the first time in orchids.

Morphological and Physio-Chemical Responses to PEG-Induced Water Stress in Vanilla planifolia and V. pompona Hybrids.

Vanilla planifolia is an orchid of cultural and economic value. However, its cultivation in many tropical countries is threatened by water stress. In contrast, V. pompona is a species that is tolerant of prolonged periods of drought. Due to the need for plants' resistant to water stress, the use of hybrids of these two species is considered. Therefore, the objective of this study was to evaluate the morphological and physio-chemical responses of in vitro vanilla seedlings of the parental genotype V. planifolia, and the hybrids V. planifolia × V. pompona and V. pompona × V. planifolia, which were then exposed over five weeks to polyethylene glycol-induced water stress (-0.49 mPa). Stem and root length, relative growth rate, number of leaves and roots, stomatal conductance, specific leaf area, and leaf water content were determined. Metabolites potentially associated with the response to water stress were identified in leaves, through untargeted and targeted metabolomics. Both hybrids exhibited a smaller decrease in the morphophysiological responses compared to V. planifolia and exhibited an enrichment of metabolites such as carbohydrates, amino acids, purines, phenols, and organic acids. Hybrids of these two species are considered as a potential alternative to the traditional cultivation of vanilla to face drought in a global warming scenario.

Free comment as a valuable approach to characterize and identify the drivers of liking of high-protein flavored milk drink submitted to ohmic heating.

Flavored milk drink is a popular dairy product traditionally processed by pasteurization, which is a safe and robust process. Still, it can imply a greater energy expenditure and a more significant sensorial alteration. Ohmic heating (OH) has been proposed as an alternative to dairy processing, including flavored milk drink. However, its impact on sensory characteristics needs to be evidenced. This study used Free Comment, an underexplored methodology in sensory studies, to characterize five samples of high-protein vanilla-flavored milk drink: PAST (conventional pasteurization 72 °C/15 s); OH6 (ohmic heating at 5.22 V/cm); OH8 (ohmic heating at 6.96 V/cm); OH10 (ohmic heating at 8.70 V/cm), and OH12 (ohmic heating at 10.43 V/cm). Free Comment raised similar descriptors to those found in studies that used more consolidated descriptive methods. The employed statistical approach allowed observation that pasteurization and OH treatment have different effects on the sensory profile of products, and the electrical field strength of OH also has a significant impact. PAST was slightly to moderately negatively associated with "acid taste," "fresh milk taste," "smoothness," "sweet taste," "vanilla flavor," "vanilla aroma," "viscous," and "white color." On the other hand, OH processing with more intense electric fields (OH10 and OH12) produced flavored milk drinks strongly associated with the "in natura" milk descriptors ("fresh milk aroma" and "fresh milk taste"). Furthermore, the products were characterized by the descriptors "homogeneous," "sweet aroma," "sweet taste," "vanilla aroma," "white color," "vanilla taste," and "smoothness." In parallel, less intense electric fields (OH6 and OH8) produced samples more associated with a bitter taste, viscosity, and lumps presence. Sweet taste and fresh milk taste were the drivers of liking. In conclusion, OH with more intense electric fields (OH10 and OH12) was promising in flavored milk drink processing. Furthermore, the free comment was a valuable approach to characterize and identify the drivers of liking of high-protein flavored milk drink submitted to OH.

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.

Structural plasticity in roots of the hemiepiphyte Vanilla phaeantha Rchb.f. (Orchidaceae): a relationship between environment and function.

The aerial environment appears to structurally modify roots, which frequently show specializations for absorbing water and nutrients. Among those specializations are the velamen, a multiseriate epidermis generally composed of dead mature cells, and greater degrees of lignification in the endodermis, exodermis, and pith. Vanilla phaeantha is a hemiepiphyte used here as a model of study to determine which root characteristics demonstrate the most plasticity in response to aerial and terrestrial environments. It produces roots growing under three conditions: (1) aerial and free, growing from the highest branches towards the ground; (2) aerial roots attached to the phorophyte; and (3) terrestrial. Samples taken 3 cm from the apices were used to prepare histological slides. The tissues and other anatomical structures were measured and histochemically characterized. The most plastic characteristics were the external periclinal thicknesses of the exodermis and the total area occupied by the aerenchyma lacunae. The free roots were the longest, did not evidence root hairs, and had the largest number of the aerenchyma lacunae; they also evidenced greater thicknesses of the exodermis in contact with the epidermis walls that helped maintain their shapes. Terrestrial roots had root hairs around the entire circumference and intense infestations of mycorrhiza, indicating their involvement in nutrient acquisition. The adhering roots evidenced free regions similar to those of aerial roots, as well as adhering regions showed characteristics similar to terrestrial roots (with root hairs and mycorrhiza infestations).

Advances in Somatic Embryogenesis in Vanilla (Vanilla planifolia Jacks.).

Somatic embryogenesis is an in vitro plant morphogenetic process due to cell totipotentiality to induce shoot regeneration. To induce this proliferation pathway, we used auxins such as 2,4-dichlorophenoxyacetic acid in combination with cytokinins. There are numerous somatic embryogenesis protocols for a great diversity of plants, including orchids, but none has been yet reported in Vanilla planifolia. Vanilla (V. planifolia) is propagated mainly asexually through cuttings. Under in vitro conditions, it is propagated asexually through direct and indirect organogenesis involving the use of various plant growth regulators in different concentrations. The cell response depends on explant type, culture medium used, and incubation conditions. Direct organogenesis involves de novo formation from differentiated cells; the indirect pathway develops from cell dedifferentiation that produces an explant called "callus." In most cases, this type of cell regeneration uses Benzyladenine. The explants most used in this pathway are shoots, roots, and protocorms, although some studies report the use of other types of explants, including leaves and seeds. Somatic embryogenesis in vanilla has been poorly studied partly because of the recalcitrance of this species. This work mentioned the advances in the in vitro morphogenesis of V. planifolia, mentioning the advantages and disadvantages of each morphogenetic pathway and its characteristics.

Uncovering haplotype diversity in cultivated Mexican vanilla species.

PREMISE: Although vanilla is one of the best-known spices, there is only a limited understanding of its biology and genetics within Mexico, where its cultivation originated and where phenotypic variability is high. This study aims to augment our understanding of vanilla's genetic resources by assessing species delimitation and genetic, geographic, and climatic variability within Mexican cultivated vanilla. METHODS: Using nuclear and plastid DNA sequence data from 58 Mexican samples collected from three regions and 133 ex situ accessions, we assessed species monophyly using phylogenetic analyses and genetic distances. Intraspecific genetic variation was summarized through the identification of haplotypes. Within the primarily cultivated species, Vanilla planifolia, haplotype relationships were further verified using plastome and rRNA gene sequences. Climatic niche and haplotype composition were assessed across the landscape. RESULTS: Three species (Vanilla planifolia, V. pompona, and V. insignis) and 13 haplotypes were identified among Mexican vanilla. Within V. planifolia haplotypes, hard phylogenetic incongruences between plastid and nuclear sequences suggest past hybridization events. Eight haplotypes consisted exclusively of Mexican samples. The dominant V. planifolia haplotype occurred throughout all three regions as well as outside of its country of origin. Haplotype richness was found to be highest in regions around Papantla and Chinantla. CONCLUSIONS: Long histories of regional cultivation support the consideration of endemic haplotypes as landraces shaped by adaptation to local conditions and/or hybridization. Results may aid further genomic investigations of vanilla's genetic resources and ultimately support the preservation of genetic diversity within the economically important crop.

Metabolite transformation and ß-d-glucosidase activity during the high hydrostatic pressure assisted curing of vanilla beans (Vanilla planifolia) to improve phenolic compounds formation.

Current methods for vanilla bean curing are long and reduce the enzymatic activity necessary for flavor development. High hydrostatic pressure (HHP) at 50-600 MPa was used to improve phenolic compounds formation and ß-d-glucosidase activity in vanilla beans compared with scalded beans. Phenolics were analyzed by HPLC and ß-d-glucosidase activity by spectrophotometry. Vanillin was the main phenolic and it was formed by ß-d-glucovanillin hydrolysis and vanillyl alcohol oxidation. HHP improved vanillin content and influenced ß-d-glucosidase activity. At the beginning of the curing the highest increments of vanillin were produced at 400 MPa (up to 15%), while at the end, this was observed at 50 (138%) and 600 MPa (74%). Maximum increment of up to 400% in ß-d-glucosidase activity was observed from 100 to 300 MPa, which was attributed to tissue decompartmentalization, and conformational changes induced by pressure. HHP could be used during vanilla curing to improve vanillin content and ß-d-glucosidase activity.

Microbial Diversity in Cultivated and Feral Vanilla Vanilla planifolia Orchids Affected by Stem and Rot Disease.

The worldwide production of vanilla, a native orchid from Mexico, is greatly affected by stem and root rot disease (SRD), typically associated with Fusarium oxysporum fungi. We hypothesized that the presence of Fusarium species in vanilla is not sufficient for the plant to express symptoms of the disease. We described the taxonomic composition of endophytic microbiomes in symptomatic and asymptomatic vanilla plants using 16S and ITS rDNA metabarcoding, and ITS Sanger sequences generated from fungal isolates. We compared the bacterial and fungal diversity in vanilla plants from a long-term plantation, and from feral plants found near abandoned plantations that did not present SRD symptoms. No significant differences were found in the species richness of the bacterial and fungal microbiome among feral, or asymptomatic and symptomatic cultivated vanilla. However, significant differences were detected in both fungal and bacterial diversity from different organs in the same plant, with roots being more diverse than stems. We found that Proteobacteria and Actinobacteria, as well as the fungal families Nectriaceae and Xylariaceae, constitute the core of the vanilla microbiome that inhabits the root and stem of both cultivated and feral plants. Our work provides information on the microbial diversity associated to root and stem rot in vanilla and lays the groundwork for a better understanding of the role of the microbiome in vanilla fungal diseases.

Evolutionary disruption in the pollination system of Vanilla (Orchidaceae).

Pollination of the pantropical Vanilla has been linked to melittophily and food deception. Here we investigated the role of flower traits on the reproduction of Neotropical Vanilla. We also studied the evolution of pollination systems in order to understand the origin of production of flower resources and the diversification of pollinators in this orchid genus. Our study was founded on data of adaptations in flower morphology, production of resources, scent release, pollinators and breeding systems of Vanilla and presenting new data on reproductive biology of V. palmarum. Data on reproductive biology of Vanilla were mapped onto a phylogeny to address our queries on the evolution of pollination systems in this genus. Vanilla palmarum shows a mixed mating system, with its facultative autogamous flowers being pollinated by hummingbirds. Its yellow flowers are scentless and produces nectar. Mapping of the pollination system onto trees resulted in one origin for bird pollination and at least two origins for autogamy in Vanilla. Nectar secretion has a single origin in the Neotropical thick-leafed lineage. Bird pollination of Vanilla is shown for the first time. The origin of ornithophily within a bee-pollinated clade is supported by flower morphology. Floral transitions to ornithophily have been favoured by the occupation of a distinct niche from that of the other thick-leafed Vanilla species. Despite its specialized pollination, V. palmarum is autogamous. A mixed mating system can promote reproductive assurance in the case of a decline in pollinator populations, or in areas where pollinator services are irregular or absent.

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.

Unravelling the enigma of seed dispersal in Vanilla.

Vanilla bean is the second most expensive spice in the world. While widely cultivated for centuries due to its importance for industry and gastronomy, its dispersers are unknown. In the present study, I investigated the system of seed dispersal of Neotropical vanillas based on experimental observations, the morpho-anatomy of fruits and seeds, and the effect of dormancy breakdown on seed germination. My results show the central cavity of the fruit contains copious amounts of seed with sclerified coats that are consumed by birds. The combination of gradually opening dehiscent fruits and a well-developed mesocarp rich in raphide idioblasts reinforces the idea that seed dispersal in Vanilla is more adapted to non-chewing animals. The digestive acids of birds sclerify the hard seed coats, breaking dormancy and promoting germination at the beginning of the wet season, which is fundamental for seedling survival and for the establishment of plants in the early stages of development during the rainy season. Zoochory in Vanilla is show for the first time. The chemical scarification of the seed coat is crucial for synchronizing the biological processes involved in seed germination. My evidence on endozoochory and the processes involved in seed germination of Neotropical vanillas provides new insights into understanding of the early evolution of seed dispersal in orchids.

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.