Exploring endophytic bacteria communities of Vanilla planifolia.

BACKGROUND: Rhizosphere bacterial community and endophytes are now known to influence plant health and response to environmental stress. Very few studies have reported the diversity of endophytic bacterial communities of Vanilla planifolia and their potential roles in promoting plant growth or contributing to aromatic quality. RESULTS: In this study, the composition and diversity of the Vanilla rhizosphere bacterial community were explored by analyzing rhizosphere soil and root tissue samples as well as green pods of three accessions of Vanilla planifolia grown on different types of substrates (compost and leaf litter). In addition, the endophytic bacterial diversity of roots and green pods as well as the evolution of endophytic bacteria after the curing process of vanilla green pods were analyzed based on a metabarcoding approach. The results showed that bacterial species richness and diversity were higher in the compost. The analysis of the soil bacterial composition displayed that Halomonas, Pseudoalteromonas, Enterobacter and Bradyrhizobium were the most abundant genera. Moreover, the results indicated that the soil bacterial community structure was linked to the host plant genotype. Regarding the roots endophytic bacteria composition, the genera Halomonas, Pseudoalteromonas, Bacillus and Carboxydocella genera were present in all samples, independently from the substrate nature. Several genera including Bacillus, Bradyrhizobium, Burkholderia and Halomonas were transmitted internally from the roots to the green pods. The curing process reduced the bacterial richness and bacterial diversity associated with the green pods. Halomonas, Pseudoalteromonas, Bacillus, and Carboxydocella are the dominant genera in the pods after the curing process. CONCLUSIONS: This study provides an overview of changes of the bacterial communities dynamics especially endophytic in the roots and the green pods. It highlighted bacterial genera (Halomonas, Pseudoalteromonas, Bacillus, and Carboxydocella) potentially implicated in the formation of aroma compounds of vanilla beans.

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.

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.

Diagnostic KASP markers differentiate Vanilla planifolia, V. odorata, V. pompona, and their hybrids using leaf or cured pod tissues.

BACKGROUND: Vanilla is a globally important spice crop used in a variety of food, cosmetic, and pharmaceutical products. V. planifolia is the primary commercial species with V. x tahitensis also permissible for food use. Other aromatic species, including V. pompona, are used for food throughout Central and South America. Supply chain complexity hinders the vanilla bean industry and can lead to false claims of genetic and geographical origins to obtain higher prices. Beans of some species can be differentiated by experienced buyers, but hybrids and morphological differences caused by environmental variability or disease would best be resolved by diagnostic tests. METHODS AND RESULTS: Kompetitive Allele Specific Polymerase Chain Reaction is a widely used molecular marker that can genotype single nucleotide polymorphisms efficiently and inexpensively. Assays were designed to differentiate V. planifolia, V. x tahitensis, and V. pompona using publicly available vanilla genomics data. Ten KASP assays on chromosomes 1 through 7, the ITS region, and plastid-encoded rbcL gene successfully differentiated V. planifolia, V. odorata, and V. x tahitensis. Additional KASP assays on chromosomes 1 through 4, the ITS region, and rbcL gene successfully differentiated V. planifolia and V. pompona. Further, a method for extracting KASP-quality DNA from cured vanilla bean seeds was developed and successfully differentiated V. planifolia, V. odorata, V. x tahitensis, V. pompona, and their hybrids. CONCLUSION: The methods and results from this study can be used to identify interspecific hybrids, ensure the authenticity of cured vanilla beans, and reduce abuse within the vanilla supply chain.

First Vanilla planifolia High-Density Genetic Linkage Map Provides Quantitative Trait Loci for Resistance to Fusarium oxysporum.

Fusarium oxysporum f. sp. radicis-vanillae (Forv), the causal agent of root and stem rot disease, is the main pathogen affecting vanilla production. Sources of resistance have been reported in Vanilla planifolia G. Jackson ex Andrews, the main cultivated vanilla species. In this study, we developed the first high-density genetic map in this species with 1,804 genotyping-by-sequencing (GBS)-generated single nucleotide polymorphism (SNP) markers using 125 selfed progenies of the CR0040 traditional vanilla cultivar. Sixteen linkage groups (LG) were successfully constructed, with a mean of 113 SNPs and an average length of 207 cM per LG. The map had a high density with an average of 5.45 SNP every 10 cM and an average distance of 1.85 cM between adjacent markers. The first three LG were aligned against the first assembled chromosome of CR0040, and the other 13 LG were correctly associated with the other 13 assembled chromosomes. The population was challenged with the highly pathogenic Forv strain Fo072 using the root-dip inoculation method. Five traits were mapped, and 20 QTLs were associated with resistance to Fo072. Among the genes retrieved in the CR0040 physical regions associated with QTLs, genes potentially involved in biotic resistance mechanisms, coding for kinases, E3 ubiquitin ligases, pentatricopeptide repeat-containing proteins, and one leucine-rich repeat receptor underlying the qFo72_08.1 QTL have been highlighted. This study should provide useful resources for marker-assisted selection in V. planifolia.

A High-Resolution Network with Strip Attention for Retinal Vessel Segmentation.

Accurate segmentation of retinal vessels is an essential prerequisite for the subsequent analysis of fundus images. Recently, a number of methods based on deep learning have been proposed and shown to demonstrate promising segmentation performance, especially U-Net and its variants. However, tiny vessels and low-contrast vessels are hard to detect due to the issues of a loss of spatial details caused by consecutive down-sample operations and inadequate fusion of multi-level features caused by vanilla skip connections. To address these issues and enhance the segmentation precision of retinal vessels, we propose a novel high-resolution network with strip attention. Instead of the U-Net-shaped architecture, the proposed network follows an HRNet-shaped architecture as the basic network, learning high-resolution representations throughout the training process. In addition, a strip attention module including a horizontal attention mechanism and a vertical attention mechanism is designed to obtain long-range dependencies in the horizontal and vertical directions by calculating the similarity between each pixel and all pixels in the same row and the same column, respectively. For effective multi-layer feature fusion, we incorporate the strip attention module into the basic network to dynamically guide adjacent hierarchical features. Experimental results on the DRIVE and STARE datasets show that the proposed method can extract more tiny vessels and low-contrast vessels compared with existing mainstream methods, achieving accuracies of 96.16% and 97.08% and sensitivities of 82.68% and 89.36%, respectively. The proposed method has the potential to aid in the analysis of fundus images.

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.

Evolutionary Patterns of the Chloroplast Genome in Vanilloid Orchids (Vanilloideae, Orchidaceae).

The Vanilloideae (vanilloids) is one of five subfamilies of Orchidaceae and is composed of fourteen genera and approximately 245 species. In this study, the six new chloroplast genomes (plastomes) of vanilloids (two Lecanorchis, two Pogonia, and two Vanilla species) were decoded, and then the evolutionary patterns of plastomes were compared to all available vanilloid plastomes. Pogonia japonica has the longest plastome, with 158,200 bp in genome size. In contrast, Lecanorchis japonica has the shortest plastome with 70,498 bp in genome size. The vanilloid plastomes have regular quadripartite structures, but the small single copy (SSC) region was drastically reduced. Two different tribes of Vanilloideae (Pogonieae and Vanilleae) showed different levels of SSC reductions. In addition, various gene losses were observed among the vanilloid plastomes. The photosynthetic vanilloids (Pogonia and Vanilla) showed signs of stage 1 degradation and had lost most of their ndh genes. The other three species (one Cyrotsia and two Lecanorchis), however, had stage 3 or stage 4 degradation and had lost almost all the genes in their plastomes, except for some housekeeping genes. The Vanilloideae were located between the Apostasioideae and Cypripedioideae in the maximum likelihood tree. A total of ten rearrangements were found among ten Vanilloideae plastomes when compared to the basal Apostasioideae plastomes. The four sub-regions of the single copy (SC) region shifted into an inverted repeat (IR) region, and the other four sub-regions of the IR region shifted into the SC regions. Both the synonymous (dS) and nonsynonymous (dN) substitution rates of IR in-cooperated SC sub-regions were decelerated, while the substitution rates of SC in-cooperated IR sub-regions were accelerated. A total of 20 protein-coding genes remained in mycoheterotrophic vanilloids. Almost all these protein genes show accelerated base substitution rates compared to the photosynthetic vanilloids. Two of the twenty genes in the mycoheterotrophic species faced strong "relaxed selection" pressure (p-value < 0.05).

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.

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.

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.

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.

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).

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.

Effects of 3-octen-2-one on human olfactory receptor responses to vanilla flavor.

Most of the odors that humans perceive daily are complex odors. It is believed that the modulation, enhancement, and suppression of overall complex odors are caused by interactions between odor molecules. In this study, to understand the interaction between odor molecules at the level of human olfactory receptor responses, the effects of 3-octen-2-one, which has been shown to modulate vanilla flavors, were analyzed using a human olfactory receptor sensor that uses all human olfactory receptors (388 types) as sensing molecules. As a result, the response intensity of 1 common receptor (OR1D2) was synergistically enhanced in vanilla flavor with 3-octen-2-one compared with vanilla flavor, and the response of 1 receptor (OR5K1) to vanilla flavor was completely suppressed. These results strongly suggested that the response of human olfactory receptors to complex odors is enhanced or suppressed by relatively few other odor molecules.

Improving Network Training on Resource-Constrained Devices via Habituation Normalization.

As a technique for accelerating and stabilizing training, the batch normalization (BN) is widely used in deep learning. However, BN cannot effectively estimate the mean and the variance of samples when training/fine-tuning with small batches of data on resource-constrained devices. It will lead to a decrease in the accuracy of the deep learning model. In the fruit fly olfactory system, the algorithm based on the "negative image" habituation model can filter redundant information and improve numerical stability. Inspired by the circuit mechanism, we propose a novel normalization method, the habituation normalization (HN). HN first eliminates the "negative image" obtained by habituation and then calculates the statistics for normalizing. It solves the problem of accuracy degradation of BN when the batch size is small. The experiment results show that HN can speed up neural network training and improve the model accuracy on vanilla LeNet-5, VGG16, and ResNet-50 in the Fashion MNIST and CIFAR10 datasets. Compared with four standard normalization methods, HN keeps stable and high accuracy in different batch sizes, which shows that HN has strong robustness. Finally, the applying HN to the deep learning-based EEG signal application system indicates that HN is suitable for the network fine-tuning and neural network applications under limited computing power and memory.

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.

Effects of Different Extraction Methods on Vanilla Aroma.

To establish the analytic conditions for examining the aroma quality of vanilla pods, we compared different extraction methods and identified a suitable option. We utilized headspace solid-phase microextraction (HS-SPME), steam distillation (SD), simultaneous steam distillation (SDE) and alcoholic extraction combined with gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) to identify volatile components of vanilla pods. A total of 84 volatile compounds were identified in this experiment, of which SDE could identify the most volatile compounds, with a total of 51 species, followed by HS-SPME, with a total of 28 species. Ten volatile compounds were identified by extraction with a minimum of 35% alcohol. HS-SPME extraction provided the highest total aroma peak areas, and the peak areas of aldehydes, furans, alcohols, monoterpenes and phenols compounds were several times higher than those of the other extraction methods. The results showed that the two technologies, SDE and HS-SPME, could be used together to facilitate analysis of vanilla pod aroma.

Simultaneous Determination of Ferulic Acid and Vanillin in Vanilla Extracts Using Voltammetric Sensor Based on Electropolymerized Bromocresol Purple.

Natural phenolic antioxidants are one of the widely studied compounds in life sciences due to their important role in oxidative stress prevention and repair. The structural similarity of these antioxidants and their simultaneous presence in the plant samples stipulate the development of methods for their quantification. The current work deals with the simultaneous determination of vanillin and its bioprecursor ferulic acid using a voltammetric sensor for the first time. A sensor based on the layer-by-layer deposition of the polyaminobenzene sulfonic acid functionalized single-walled carbon nanotubes (f-SWCNTs) and electropolymerized bromocresol purple has been developed for this purpose. The best response of co-existing target analytes was registered for the polymer obtained from the 25 µM dye by 10-fold potential cycling from 0.0 to 1.2 V with the scan rate of 100 mV s-1 in 0.1 M phosphate buffer (PB), pH 7.0. Scanning electron microscopy (SEM), cyclic voltammetry and electrochemical impedance spectroscopy (EIS) confirmed the effectivity of the sensor developed. The linear dynamic ranges of 0.10-5.0 µM and 5.0-25 µM for both analytes with the detection limits of 72 nM and 64 nM for ferulic acid and vanillin, respectively, were achieved in differential pulse mode. The sensor was applied for the analysis of vanilla extracts.

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.