Astringency Sensitivity to Tannic Acid: Effect of Ageing and Saliva.

Astringency is an important sensory characteristic of food and beverages containing polyphenols. However, astringency perception in elderly people has not been previously documented. The aim of the present work was to evaluate sensitivity to astringency as a function of age, salivary flow and protein amount. Fifty-four panellists, including 30 elderly people (age = 75 ± 4.2 years) and 24 young people (age = 29.4 ± 3.8 years), participated in this study. Astringency sensitivity was evaluated by the 2-alternative forced choice (2-AFC) procedure using tannic acid solutions. Whole saliva was collected for 5 min before and after the sensory tests. The results showed that the astringency threshold was significantly higher in the elderly group than the young group. No correlation was observed between the salivary protein amount and threshold value. However, a negative correlation between salivary flow and threshold was observed in the young group only. These results showed a difference in oral astringency perception as a function of age. This difference can be linked to salivary properties that differ as a function of age.

The association between changes of gustatory function and changes of salivary parameters: A pilot study.

OBJECTIVE: The aim of the pilot study was to explore which of the salivary parameters best reflects improvement or deterioration of taste function. METHODS: A total of 14 patients were included. Taste ability was measured using taste strips and patients rated their symptom strength using visual analogue scales. Salivary parameters (flow rate, total proteins, proteolysis, catalase, total anti-oxidative capacity [TAC], carbonic anhydrase VI [caVI], and pH) were determined and the Beck Depression Inventory (BDI) was administered. All these parameters were measured twice with a one-year interval to acquire the changes of data. RESULTS: Patients with decreased taste function exhibited a decrease in salivary proteolysis and caVI, and an increase in salivary total protein. Patients with increased taste function also showed an increase in salivary total protein. Δ Salivary flow rate was negatively correlated with Δ taste strip scores. Δ Salivary pH was significantly lower in patients with increased taste function compared to patients with decreased taste function. Δ BDI was positively correlated with both Δ symptoms ratings. Across all patients, symptom ratings decreased while salivary total protein increased; salivary flow rate, proteolysis and caVI decreased significantly compared with baseline. CONCLUSIONS: The present longitudinal results suggest that changes of both taste function and taste complaints were accompanied by changes in salivary parameters, indicating that salivary parameters have the potential to be useful in the diagnosis of patients with qualitative taste disorders.

Longitudinal analysis of the salivary metabolome of breast-fed and formula-fed infants over the first year of life.

INTRODUCTION: The salivary metabolome has been increasingly studied over the past ten years due to the potential of saliva as a non-invasive source of biomarkers. However, although saliva has been studied in relation to various diseases, its dynamic evolution during life is not known. This is particularly true for the first months of life. Infancy is indeed a critical period during which numerous behavioural and physiological events occur, such as dietary transitions and tooth eruption, which can lead to important biological modifications in the oral cavity. OBJECTIVES: The aim of this work was therefore to study the evolution of the salivary metabolome during the first months of life by 1H NMR. METHODS: Saliva of 32 infants with different milk feeding histories (breast vs formula) was collected at 6 stages, including 3 months old, 15 days before the onset of complementary feeding (CF), approximately 15 days after the onset of CF, approximately 21 days after the onset of CF and at approximately 11 and 15 months, and analysed. RESULTS: The longitudinal analysis showed a significant modification of the profiles of 18 metabolites over time; 14 presented an increase in abundance whereas 4 presented a decrease. These modifications seemed to be linked, for the most part, to an increase in oral microbial metabolism. Milk feeding history during the first months of life had no effect on metabolites. CONCLUSION: This work shows that the salivary metabolome should be considered when studying the changes occurring during infancy.

Acceptance of added fat to first complementary feeding purees: An exploration of fat type, feeding history and saliva composition.

In adults, fat is a major determinant of food palatability. From the onset of complementary feeding (CF) adding fat to complementary foods is recommended to ensure an optimal growth and cognitive development. However, whether adding fat to complementary foods would impact acceptance (in terms of intake and liking) has been little investigated. This study sought 1) to evaluate acceptance of added fat (either vegetable oils or dairy fat) in a vegetable puree in weaning-age infants; 2) to determine whether early differential fat exposure through milk (breast milk and formula have different fat composition) and fat addition in complementary foods can influence acceptance and 3) to explore if fat acceptance can be related to inter-individual differences in salivary compounds potentially involved in fat perception. Twenty six infants with contrasted milk feeding history participated and were introduced with complementary foods at 4.8 months. During the 1st month of complementary feeding, acceptance of 3 broccoli purees (0% fat, 7% of vegetable oils, 7% of dairy fat) was determined through ad libitum intake and global liking, in the laboratory and at home. Saliva was collected: lipolytic activity and carbonic anhydrase 6 concentration were determined. Puree intakes were not impacted by fat addition, whatever the type of added fat. Moreover, the history of milk feeding (breast milk vs. vegetable oils based formulas) in the very first months did not explain acceptance for added fat. Finally, no links between intake and saliva composition were evidenced. Altogether, this study found that the addition of fat did not modify food acceptance by infants during early complementary feeding. Thus, future research should investigate the development of fat acceptance over experience in early infancy.

Differences in the Density of Fungiform Papillae and Composition of Saliva in Patients With Taste Disorders Compared to Healthy Controls.

This study investigated the relation of the fungiform taste papillae density and saliva composition with the taste perception of patients suffering from diagnosed taste disorders. For this purpose, 81 patients and 40 healthy subjects were included. Taste was measured by means of regional and whole mouth chemosensory tests, and electrogustometry. Olfaction was assessed using the Sniffin Sticks. Fungiform papillae were quantified using the "Denver Papillae Protocol for Objective Analysis of Fungiform Papillae". In addition, salivary parameters [flow rate, total proteins, catalase, total anti-oxidative capacity (TAC), carbonic anhydrase VI (caVI), and pH] were determined and the Beck Depression Inventory was administered. Patients showed less taste papillae compared to healthy subjects. The number of papillae correlated with total taste strip score and salivary flow rate. Regarding salivary parameters, the flow rate, protein concentration, and TAC of patients were higher compared to controls. In addition, salivary flow rate, protease, caVI, and catalase values correlated with the summed taste strip score. Regarding various taste disorders, salty-dysgeusia patients showed the lowest taste test scores compared to those with bitter or metal-dysgeusia. Olfactory function of patients was significantly worse compared to healthy controls. This difference was most pronounced for ageusia patients. Compared to controls, patients also exhibited higher depressive symptoms. The density of fungiform papillae seemed to be positively associated with taste perception. Furthermore, patients exhibited changes in saliva composition (higher salivary flow rate, increased protein concentration, proteolysis, and TAC) compared to controls indicating that assessment of saliva may be critical for the diagnostic procedure in taste disorders.

Astringency sensitivity to tannic acid: Effect of ageing and salivary proline-rich protein levels.

The link between salivary composition and sensitivity to astringency as a function of age has still not been established. In this work, we propose the hypothesis that ageing leads to changes in the concentration of salivary proline-rich proteins (PRPs), which alters the astringency perception threshold with age. To test this hypothesis, astringency sensitivity to tannic acid and saliva was assessed in 30 elderly people and 24 young people. Basic PRPs (bPRPs) and glycosylated PRPs (gPRPs) were quantified immunochemically via western blot analysis. The results showed that the amounts of bPRPs and gPRPs were similar between the young and elderly groups. However, a positive correlation between the gPRP amount and astringency threshold was observed only in the young group, while a negative correlation between the bPRP amount and astringency threshold was observed only in the elderly group. This finding suggests differences in the contribution of PRP type to astringency perception as a function of age.

Development of New Models of Oral Mucosa to Investigate the Impact of the Structure of Transmembrane Mucin-1 on the Mucosal Pellicle Formation and Its Physicochemical Properties.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

Role of human salivary enzymes in bitter taste perception.

The molecules that elicit taste sensation are perceived by interacting with the taste receptors located in the taste buds. Enzymes involved in the detoxification processes are found in saliva as well as in type II cells, where taste receptors, including bitter taste receptors, are located. These enzymes are known to interact with a large panel of molecules. To explore a possible link between these enzymes and bitter taste perception, we demonstrate that salivary glutathione transferases (GSTA1 and GSTP1) can metabolize bitter molecules. To support these abilities, we solve three X-ray structures of these enzymes in complexes with isothiocyanates. Salivary GSTA1 and GSTP1 are expressed in a large panel of subjects. Additionally, GSTA1 levels in the saliva of people suffering from taste disorders are significantly lower than those in the saliva of the control group.

Perspectives on Astringency Sensation: An Alternative Hypothesis on the Molecular Origin of Astringency.

Flavor is one of the main drivers of food consumption and acceptability. It is associated with pleasure feels during eating. Flavor is a multimodal perception corresponding to the functional integration of information from the chemical senses: olfaction, gustation, and nasal and oral somatosensory inputs. As a result, astringency, as a sensation mediated by the trigeminal nerves, influences food flavor. Despite the importance of astringency in food consumer acceptance, the exact chemosensory mechanism of its detection and the nature of the receptors activated remain unknown. Herein, after reviewing the current hypotheses on the molecular origin of astringency, we proposed a ground-breaking hypothesis on the molecular mechanisms underpinning this sensation as a perspective for future research.

Proteomic characterization of the mucosal pellicle formed in vitro on a cellular model of oral epithelium.

The oral mucosal pellicle is a thin lubricating layer generated by the binding of saliva proteins on epithelial oral cells. The protein composition of this biological structure has been to date studied by targeted analyses of specific salivary proteins. In order to perform a more exhaustive proteome characterization of pellicles, we used TR146 cells expressing or not the transmembrane mucin MUC1 and generated pellicles by incubation with human saliva and washing to remove unbound proteins. A suitable method was established for the in vitro isolation of the mucosal pellicle by "shaving" it from the cells using trypsin. The extracts, the washing solutions and the saliva used to constitute the pellicles were analyzed by LC MS/MS (data are available via ProteomeXchange with identifier PXD017268). Comparison of pellicle and saliva compositions evidenced the adsorption of proteins not previously reported as pellicle constituents such as proteins of the PLUNC family. Pellicles formed on TR146 and TR146/MUC1 were also analyzed and compared by protein label-free quantification. The two types of samples appeared as distinct clusters in multivariate analyses, but the discriminant proteins (Welch test p < .05, FDR < 0.1) were cellular rather than salivary proteins. SIGNIFICANCE: The oral mucosal pellicle is made of salivary proteins tightly bound to oral epithelial cells. It is essential to oral health, with biological functions depending largely on its protein constituents. Characterizing its proteome is difficult due to the intimate association of this protein layer to cell membranes. In this work, we report a trypsin "shaving" protocol which enabled to sample the pellicle formed on an in vitro cellular model of oral epithelium. Analyzing such samples by high-resolution mass spectrometry provided novel information on the mucosal pellicle composition. This work is therefore a good starting point for further characterization of this biological structure.

Oral lipolysis and its association with diet and the perception and digestion of lipids: A systematic literature review.

OBJECTIVES: This systematic literature review aims to summarize the existing scientific evidence on the association of oral lipolysis with diet and with the perception and digestion of lipids in humans and rodents. METHODS: A validated search strategy of two databases (PubMed and ISI Web of Knowledge) was carried out and the contents were screened by two independent reviewers. The quality of the included studies was critically evaluated on the basis of the Quality Assessment Criteria for Evaluating Primary Research Papers. RESULTS: From the originally identified studies (n = 2295), 17 articles met the eligibility criteria for inclusion in the analysis. Among them, only 6 articles received the maximum assessment score. The main reason for this finding was the absence of a control for the confounding bias between lipases and esterases. In rodents, oral lipolysis was principally due to the activity of lingual lipase, which was associated with the 3 selected parameters. In humans, the association parameters were principally established through indirect evidence without a clear demonstration of cause. Moreover, no specific lipase, such as lingual lipase in the case of rodents, was identified at the oral level. CONCLUSIONS: Future research efforts should focus on (i) establishing a standard procedure for oral lipolytic activity evaluation and, in particular, a methodological control to address the lipase vs esterase confounding bias and (ii) identifying the main lipases that contribute to the lipolytic activity in humans at the oral level and their respective contribution to the association parameters defined in this review.

How are macronutrient intake, BMI, ethnicity, age, and gender related to the composition of unstimulated saliva? A case study.

This study investigated how macronutrient intake, BMI, ethnicity, age, and gender are related to the composition of unstimulated saliva. First, two groups of Caucasian, Dutch subjects varying in daily intake of carbohydrate, fat, and protein were selected. The daily intake of macronutrients differed by two- to threefold between the low (n = 14) and high (n = 16) macronutrient intake groups. The same subjects were divided into two groups based on BMI: normal weight (n = 14, 22.5 ± 2.0 kg/m2 ) and overweight (n = 16, 28.1 ± 3.4 kg/m2 ). Second, one group of Caucasian, Dutch (n = 15) and one group of Asian, Chinese (n = 15) subjects were selected. Unstimulated saliva was collected from all groups. Protein concentration, amylolytic activity, lipolytic activity, and saliva flow rate were determined. None of the salivary parameters varied according to macronutrient intake and BMI. An effect of ethnicity on protein concentration was observed (p < .01; η2 = 0.142), with Asians having a 45% higher protein concentration in unstimulated saliva than Caucasians. Age had a significant effect on all salivary parameters. Protein concentration (p < .01; η2 = 0.256), amylolytic activity (p < .01; η2 = 0.234), and lipolytic activity (p < .05; η2 = 0.207) increased with age, while saliva flow rate decreased (p < .01; η2 = 0.262). Gender had a significant effect on saliva flow rate (p < .01; η2 = 0.130), with male subjects having a 32% higher flow rate than females. Age was the factor that had the greatest impact on the characteristics of unstimulated saliva. As the modulation of saliva composition according to diet has been reported previously, the extent to which macronutrient intake can affect saliva composition needs to be further investigated. PRACTICAL APPLICATIONS: Saliva plays an important role in food oral processing. From the breakdown of food structures to the binding of flavor compounds and the formation of a swallowable bolus, saliva is essential for the perception and appreciation of foods. Identifying the factors that affect saliva composition is, therefore, necessary to understand the differences in eating behavior, food perception, and preference across different consumer groups. This article aims to highlight the importance of considering saliva variability when designing food products that meet the needs of specific consumer groups.

Protein expression in submandibular glands of young rats is modified by a high-fat/high-sugar maternal diet.

OBJECTIVE: Maternal diet has consequences on many organs of the offspring, but salivary glands have received little attention despite the importance of the saliva secretory function in oral health and control of food intake. The objective of this work was therefore to document in rats the impact of maternal high-fat/high-sugar diet (Western Diet) on submandibular glands of the progeny. DESIGN: Sprague-Dawley rat dams were fed either a Western diet or control diet during gestation and lactation and their pups were sacrificed 25 days after birth. The pups' submandibular gland protein content was characterized by means of 2D-electrophoresis followed by LC-MS/MS. Data were further analyzed by Gene Ontology enrichment analysis and protein-protein interactions mapping. The expression of two specific proteins was also evaluated using immunohistochemistry. RESULTS: Combining both male and female pups (n = 18), proteome analysis revealed that proteins involved in protein quality control (e.g. heat shock proteins, proteasome sub-units) and microtubule proteins were over-expressed in Western diet conditions, which may translate intense metabolic activity. A cluster of proteins controlling oxidative stress (e.g. Glutathione peroxidases, peroxiredoxin) and enhancement of the antioxidant activity molecular function were also characteristic of maternal Western diet as well as under-expression of annexin A5. The down-regulating effect of maternal Western diet on Annexin A5 expression was significant only for males (p < 0.05). CONCLUSIONS: A maternal Western diet modifies the protein composition of the offspring's salivary glands, which may have consequences on the salivary function.

Obese Subjects With Specific Gustatory Papillae Microbiota and Salivary Cues Display an Impairment to Sense Lipids.

Some obese subjects overeat lipid-rich foods. The origin of this eating behavior is unknown. We have here tested the hypothesis that these subjects could be characterized by an impaired fatty taste sensitivity linked to a change in the gustatory papillae microbial and salivary environment. The composition of microbiota and saliva surrounding the circumvallate papillae was analyzed in combination with the orosensory lipid detection threshold in normal weight (NW) and obese (O) adults. Microbial architecture was similar to what was known in feces, but with an increased frequency of Proteobacteria. No difference in the orosensory sensitivity to lipids and composition of oral microbiota and saliva was observed between NW and O subjects. By contrast, specific bacterial and salivary signatures were found in lipid non-tasters, irrespectively of BMI. A multivariate approach highlighted that the salivary flow, lysozyme activity, total antioxidant capacity and TM7 bacterial family discriminated between tasters and non-tasters. Subgroup analysis of obese tasters (OT) versus obese non-tasters (ONT) identified specific bacterial metabolic pathways (i.e. phosphotransferase and simple sugar transport systems) as being higher in ONT. Altogether with the identification of a set of significant salivary variables, our study suggests that an "obese tongue" phenotype is associated with decreased orosensory sensitivity to lipids in some obese subjects.

Author Correction: Obese Subjects With Specific Gustatory Papillae Microbiota and Salivary Cues Display an Impairment to Sense Lipids.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The basal free fatty acid concentration in human saliva is related to salivary lipolytic activity.

Fat perception during eating is a complex sensation that involves various sensory modalities, such as texture, aroma and taste. Taste is supported by the discovery of fatty acid receptors in the tongue papillae. Dietary fat is mainly composed of esterified fatty acids, whereas only free fatty acids can bind to taste receptors. Some authors have mentioned the necessity and efficiency of salivary lipolytic activity to hydrolyse the esterified fatty acids present in foods and enable fat perception. Our hypothesis is that salivary lipolytic activity is also involved in regulating the basal level of salivary fatty acids in humans. To test this hypothesis, total fatty acid (TFA) and free fatty acid (FFA) concentrations and selected salivary characteristics (such as lipolytic activity) were analysed in the resting saliva of 54 subjects. The results show differences in the TFA and FFA profiles, with TFA and FFA concentrations of 8.99 and 3.56 µg/mL of saliva, respectively. Interestingly, lipolytic activity had a significant positive correlation with FFA concentration (0.51, p < 0.01). This result highlights a possible physiological role of salivary lipolytic activity in the regulation of the basal FFA concentration. This regulation could be involved in fat taste sensitivity.

Chewing bread: impact on alpha-amylase secretion and oral digestion.

During chewing, saliva helps in preparing the food bolus by agglomerating the formed particles, and it initiates enzymatic food breakdown. However, limited information is actually available on the adaptation of saliva composition during the oral processing of complex foods, especially for foods that are sensitive to salivary enzymes. We addressed this question in the context of starch-based products and salivary alpha-amylase. The objectives were two-fold: (1) to determine if salivary alpha-amylase secretion can be modulated by the bread type and (2) to evaluate the contribution of the oral phase in bread enzymatic breakdown. Mouthfuls of three different wheat breads (industrial, artisan and whole-meal breads) were chewed by twelve subjects. Saliva samples were collected at rest and at different times corresponding to 33, 66 and 100% of the individual's chewing sequence. Alpha-amylase activity and total protein content were determined for all saliva samples that were collected. Additionally, the salivary maltose concentration was measured as a marker of bread enzymatic digestion. Boluses were collected at the swallowing time to evaluate the saliva uptake. Chewing industrial bread induced higher saliva uptake than the other breads despite a similar chewing duration. The evolution of salivary amylase activity tended to depend on the type of bread and was highly influenced by a large degree of inter- and intra-subject variability. The protein and maltose concentration steadily increased during chewing as a result of bread breakdown. The salivary protein concentration was mainly affected by the release of the water-soluble proteins of the bread. The salivary maltose concentration was found to be significantly lower for the whole-meal bread. When considering the weight of the mouthful, enzymatic breakdown was found to be most efficient for the breads ranking from industrial > artisan > whole-meal.

Associations between food consumption patterns and saliva composition: Specificities of eating difficulties children.

Identifying objective markers of diet would be beneficial to research fields such as nutritional epidemiology. As a preliminary study on the validity of using saliva for this purpose, and in order to explore the relationship between saliva and diet, we focused on clearly contrasted groups of children: children with eating difficulties (ED) receiving at least 50% of their energy intake through artificial nutrition vs healthy controls (C). Saliva of ED and C children was analyzed by various methods (targeted biochemical analyses, 2-D electrophoresis coupled to MS, 1H NMR) and their diet was characterized using food frequency questionnaires, considering 148 food items grouped into 13 categories. Complete datasets were obtained for 16 ED and 16 C subjects (median age 4.7y and 5.0y, respectively) and the statistical link between salivary and dietary characteristics was studied by Multiple Factor Analysis (MFA). Overall, ED children showed as expected lower consumption frequency scores and higher food selectivity. The two groups of children differed in "diet/saliva" associations. Some distinctive salivary variables were common to both groups of children. For example, carbonic anhydrase 6 and the consumption frequency of biscuits & sweets and drinks were positively associated with the MFA axis 1 in C children, but oppositely associated in ED children. Specifically for ED children, abundant salivary proteins (cystatins, amylase, amylase fragments) and some metabolites (amino acids, galactose, lactate) correlated with axis 1, together with the consumption frequency of sauces & seasonings, bread & cereal products, ready-to-eat meals, fish, biscuits & sweets, drinks and potatoes. Specifically for C children, several proteins (serum albumin, haptoglobin, Igκ, apolipoprotein A-1, α-1 antitrypsin) correlated with axis 1, together with the consumption frequency of biscuits & sweets, milk & dairy products, drinks, fruit, meat and vegetables. This study demonstrates that the qualitative aspect of diet is linked to saliva composition, and that the associations between dietary consumption and salivary composition differ between groups of subjects with contrasted diets.

Multi-omics profiling reveals that eating difficulties developed consecutively to artificial nutrition in the neonatal period are associated to specific saliva composition.

Prolonged enteral or parenteral nutrition in neonatal periods sometimes results in eating difficulties persisting for years, with reduced food intake through the oral route and thereby reduced stimulation of the oral cavity. Aiming at describing the consequences on oral physiology, saliva of 21 children with eating difficulties (ED) was compared to that of 23 healthy controls, using various omics and targeted methods. Overall, despite heterogeneity within the groups (age, medication etc.), the three spectral methods (MALDI-TOF, SELDI-TOF, (1)H NMR) allowed discriminating ED and controls, confirming that oral stimulation by food intake plays a role in shaping the composition of saliva. Saliva of ED patients exhibited a lower antioxidant status and lower levels of the salivary protease inhibitors cystatins. Other discriminant features (IgA1, dimethylamine) may relate to modified oral and/or intestinal microbial ecology. Finally, salivary profiles of ED patients were partly comparable to those of subjects with exacerbated gustatory sensitivities, in particular with reduced abundance of cystatin SN and higher abundance of zinc-alpha-2-glycoprotein. Whether this translates taste hypersensitivity and contributes to the eating difficulties deserves further attention.

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells.

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner.