Molecular mechanisms of aroma persistence: From noncovalent interactions between aroma compounds and the oral mucosa to metabolization of aroma compounds by saliva and oral cells.

The present study aims to reveal the molecular mechanisms underlying aroma persistence, as it plays a major role in food appreciation and quality. A multidisciplinary approach including ex vivo experiments using a novel model of oral mucosa and saliva as well as in vivo dynamic instrumental and sensory experiments was applied. Ex vivo results showed a reduction in aroma release between 7 and 86% in the presence of the thin layer of salivary proteins covering the oral mucosa (mucosal pellicle). This reduction was explained by hydrophobic interactions involving the mucosal pellicle and by the ability of oral cells and saliva to metabolize specific aroma compounds. The in vivo evaluation of exhaled air and perception confirmed the ex vivo findings. In conclusion, this work reveals the need to consider physiological reactions occurring during food oral processing to better understand aroma persistence and open new avenues of research.

Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line.

Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor-ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.

Influence of Prebiotic Fructans on Retronasal Aroma from Elderly Individuals.

This study investigates for the first time the role of fructans with prebiotic effects (oligofructose and inulin) on retronasal aroma among elderly individuals. The impact of oligofructose (20% w/w) on retronasal aroma release was investigated using proton transfer reaction-mass spectrometry (PTR-MS) after 73 elderly individuals consumed aqueous solutions aromatized with five aroma compounds (pentan-2-one, nonan-2-one, hexan-2,3-dione, octanal and linalool). The influence of oligofructose and inulin (10% w/w) on the perceived intensity (n = 26) of two aroma descriptors (butter and floral) was also studied together with the possibility of a dumping effect on aroma evaluation due to the sweetness provided by the fructans. The results showed that the presence of oligofructose produced a significant reduction in retronasal aroma release, which could be generally explained by the physicochemical properties of aroma compounds. The presence of prebiotic fructans did not significantly affect the perceived intensity of butter and floral notes, although a dumping effect for the butter descriptor in the presence of oligofructose was observed. To conclude, these findings suggest that although fructans can exert an impact on retronasal aroma, they can be used at precise concentrations to increase the prebiotic fibre content of food products without affecting the aroma profile of foods.

Bacterial expression and purification of vertebrate odorant-binding proteins.

Vertebrate odorant-binding proteins (OBPs) are small soluble proteins abundantly secreted in the olfactory mucus of many animal species, including humans. Vertebrate OBPs reversibly bind odorant molecules with micromolar range affinities. Although their physiological role is not clearly understood, OBPs are proposed to carry airborne odorants toward membrane olfactory receptors through the nasal mucus. Measurements of odorant-OBP interactions and structural studies require a large amount of pure OBPs devoid of ligands. The bacterial expression system is the first choice for expressing vertebrate OBPs used in our laboratory and others. This system generally produces OBPs in large amounts without major problems. In this chapter, we describe the milligram-scale production of recombinant pig OBP1 (pOBP1) in E. coli. The different steps of expression and purification are presented and discussed. Protocols for secondary structures investigation by circular dichroism and binding properties of the recombinant protein are also provided. More generally, these approaches can be used to produce and characterize any vertebrate OBPs for use in functional and structural studies.

Understanding retention and metabolization of aroma compounds using an in vitro model of oral mucosa.

The mechanism leading to aroma persistence during eating is not fully described. This study aims at better understanding the role of the oral mucosa in this phenomenon. Release of 14 volatile compounds from different chemical classes was studied after exposure to in vitro models of oral mucosa, at equilibrium by Gas-Chromatography-Flame Ionization Detection (GC-FID) and in dynamic conditions by Proton Transfer Reaction- Mass Spectrometry (PTR-MS). Measurements at equilibrium showed that mucosal hydration reduced the release of only two compounds, pentan-2-one and linalool (p < 0.05), and suggested that cells could metabolize aroma compounds from different chemical families (penta-2,3-dione, trans-2-hexen-1-al, ethyl hexanoate, nonan- and decan-2-one). Dynamic analyses for pentan-2-one and octan-2-one evidenced that the constituents of the mucosal pellicle influenced release kinetics differently depending on molecule hydrophobicity. This work suggests that mucosal cells can metabolize aroma compounds and that non-covalent interactions occur between aroma compounds and oral mucosa depending on aroma chemical structure.

Nanoscale Mapping of the Physical Surface Properties of Human Buccal Cells and Changes Induced by Saliva.

The mucosal pellicle, also called salivary pellicle, is a thin biological layer made of salivary and epithelial constituents, lining oral mucosae. It contributes to their protection against microbiological, chemical, or mechanical insults. Pellicle formation depends on the cells' surface properties, and in turn the pellicle deeply modifies such properties. It has been reported that the expression of the transmembrane mucin MUC1 in oral epithelial cells improves the formation of the mucosal pellicle. Here, we describe an approach combining classical and functionalized tip atomic force microscopy and scanning microwave microscopy to characterize how MUC1 induces changes in buccal cells' morphology, hydrophobicity, and electric properties to elucidate the physicochemical mechanisms involved in the enhancement of the anchoring of salivary proteins. We show that MUC1 expression did not modify drastically the morphology of the epithelial cells' surface. MUC1 expression, however, resulted in the presence of more hydrophobic and more charged areas at the cell surface. The presence of salivary proteins decreased the highest attractive and repulsive forces recorded between the cell surface and a functionalized hydrophobic atomic force microscopy (AFM) tip, suggesting that the most hydrophobic and charged areas participate in the binding of salivary proteins. The cells' dielectric properties were altered by both MUC1 expression and the presence of a mucosal pellicle. We finally show that in the absence of MUC1, the pellicle appeared as a distinct layer poorly interacting with the cells' surface. This integrative AFM/scanning microwave microscopy approach may usefully describe the surface properties of various cell types, with relevance to the bioadhesion or biomimetics fields.

Does interindividual variability of saliva affect the release and metabolization of aroma compounds ex vivo? The particular case of elderly suffering or not from hyposalivation.

The aim of this work was to study the effects of interindividual variability of human elderly saliva on aroma release and metabolization by ex vivo approaches. Thirty individuals suffering or not from hyposalivation were selected from a panel formed by 110 elderly people (aged >65 years old) that were matched by age and sex. Then, their stimulated saliva samples were independently incubated in presence of three aroma compounds (ethyl hexanoate, octanal, 2-nonanone) to perform headspace-gas chromatography and liquid/liquid extraction-gas chromatography mass spectrometry analyses. These assays revealed that the extent of saliva effect on the release and metabolization of aroma compounds was highly dependent on the chemical family of the compounds (octanal>ethyl hexanoate>2-nonanone). Moreover, salivas from the hyposalivator (HPS) group exerted a significant lower release and/or higher metabolization than those of the control group for the three assayed compounds. Regarding the biochemical characterization of the saliva samples, no significant differences were found in the total protein content between the two groups. This does not preclude the involvement of specific proteins on the observed results that need to be clarified in further experiments. Saliva from the HPS group presented a significantly higher total antioxidant capacity than that of the control group, which suggests that this parameter could be related to the metabolization of aroma compounds by saliva. Such effects might alter aroma perception in individuals suffering from hyposalivation. PRACTICAL APPLICATIONS: The world population is getting older so fast that most countries are not prepared to handle this demographic challenge, characterized by an increasing prevalence of noncommunicable chronic diseases (e.g., diabetes, gastrointestinal disorders) associated to inadequate eating patterns. Thus, supporting a balanced diet is one of the most cost-effective strategies to maintain a good quality of life. A suitable diet needs to take into account both, specific sensory and nutritional individual needs. However, aging is often accompanied by deterioration in oral health (e.g., low salivary secretions), which could alter the capacities to taste and smell. Results from this work contribute to a better understanding of the role of human saliva in aroma release and metabolization, a first step to comprehend retronasal aroma release and perception. This knowledge will help to propose innovative solutions for the reformulation of food products better adapted to the elderly's needs, thus allowing delayed onset of dependency.

Understanding the release and metabolism of aroma compounds using micro-volume saliva samples by ex vivo approaches.

This study investigated the behaviour of key aroma compounds in the presence of human saliva (200µL) from different individuals (n=3) submitted or not to centrifugation (whole vs clarified saliva). HS-GC results showed that human saliva strongly decreased the release of carbonyl compounds (aldehydes and ketones). This effect was dependent on i) the structure of the aroma compounds and ii) the saliva composition. Whole saliva exerted a higher effect than clarified saliva on aroma compounds. Moreover, this effect was individual-dependent and related to the total protein content and the total antioxidant capacity of saliva. HS-SPME and LLE-GC/MS analyses revealed that metabolism of the compounds by salivary enzymes was involved. This observation indicates that some aroma compounds could be metabolized in the oral cavity in an individual manner, which could have implications for aroma perception (e.g., formation of new metabolites with different odor thresholds and qualities) and/or organisms' health status (e.g., compound detoxification).