# A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy

Arnaud Bernard, Loredana Radoï, Jeffrey Christensen, Florence Servant, Vincent Blasco-Blaque, Séverine Ledoux, Xavier Collet, Philippe Besnard

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## To cite this version:

Arnaud Bernard, Loredana Radoï, Jeffrey Christensen, Florence Servant, Vincent Blasco-Blaque, et al. A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy. Frontiers in Nutrition, 2023, 9, 10.3389/fnut.2022.1046454. hal-03974596

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EDITED BY

Clara G. De Los Reyes-Gavilan,
Spanish National Research Council
(CSIC), Spain

REVIEWED BY

Carolina Muñoz-Gonzalez,
Spanish National Research Council
(CSIC), Spain
Kristina Brooke Martinez-Guryn,
Midwestern University, United States

*CORRESPONDENCE

Philippe Besnard
pbesnard@u-bourgogne.fr

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This article was submitted to
Nutrition and Microbes,
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Frontiers in Nutrition

RECEIVED 16 September 2022

ACCEPTED 29 November 2022

PUBLISHED 11 January 2023

CITATION

Bernard A, Radoi L, Christensen J, Servant F, Blasco-Blaque V, Ledoux S, Collet X and Besnard P (2023) A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy.
*Front. Nutr.* 9:1046454.
doi: 10.3389/fnut.2022.1046454

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© 2023 Bernard, Radoi, Christensen, Servant, Blasco-Blaque, Ledoux, Collet and Besnard. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy

Arnaud Bernard¹, Loredana Radoi², Jeffrey Christensen³,
Florence Servant⁴, Vincent Blasco-Blaque³,
Séverine Ledoux⁵, Xavier Collet³ and Philippe Besnard¹*

¹UMR 1231 INSERM/Univ Bourgogne Franche-Comté/AgroSup Dijon, Dijon, France, ²Médecine Bucco-dentaire, Hôpital Louis Mourier (APHP-Nord), Colombes, France & Univ Paris Cité/CESP-UMR 1018 INSERM, Paris, France, ³UMR 1297 INSERM/Univ Toulouse III Paul Sabatier, Toulouse, France, ⁴Vaiomer, Toulouse, France, ⁵Explorations Fonctionnelles, Hôpital Louis Mourier (APHP-Nord), Colombes, France

**Introduction:** A preferential consumption of low-fat foods is reported by most of the patients after a vertical sleeve gastrectomy (VSG). The fact that a recent study shed light on a relationship between oral microbiota and fat taste sensitivity in obese patients prompted us to explore whether such a connection also exists in the context of a VSG.

**Methods:** Thirty-two adult female patients with a severe obesity (BMI = 43.1 ± 0.7 kg/m²) and candidates for a VSG were selected. Oral microbiota composition surrounding the gustatory circumvallate papillae (CVP) and the lipid perception thresholds were explored before and 6 months after surgery.

**Results:** VSG was found to be associated both with a qualitative (compositional changes) and quantitative (lower gene richness) remodeling of the peri-CVP microbiota. Analysis of the lipid perception allowed us to distinguish two subgroups: patients with a post-operative improvement of the fat taste sensitivity (i.e., with a lower threshold, n = 14) and unimproved patients (n = 18). Specific peri-CVP microbiota signatures also discriminated these two subgroups, unimproved patient being characterized by higher levels of *Porphyromonas*, *Fusobacterium*, and *Haemophilus* genera associated with lower levels of *Atopobium* and *Prevotella* genera as compared to the lipid-improved patients.

**Conclusion:** Collectively, these data raise the possibility that the microbial environment surrounding gustatory papillae might play a role in the positive changes of fat taste sensitivity observed in some patients after VSG.

KEYWORDS

fat taste, bariatric surgery, oral microbiota, circumvallate papillae, eating behavior
Introduction

Obesity is associated with multiple metabolic disorders at the origin of serious co-morbidities including type-2 diabetes, hypertension, or cancer. Recent changes in our food supply with, among other things, an easy access to low-cost tasty energy-dense foods rich in fat and sugar is thought to be one of major environmental contributors to the dramatic rise of obesity prevalence worldwide. A preferential consumption of high palatable foods (i.e., pleasant in mouth), rich in fat is also frequently observed in obese subjects (1-3). This eating behavior suggests that obesity *per se* might also induce food mouthfeel changes able to modify the food choice habits. An implication of the gustatory pathway in this eating pattern change is likely. Indeed, obesity alters gene expression in human taste buds (4) and reduces the abundance of gustatory fungiform papillae in mice and humans (5). Moreover, functional alterations of brain areas involved both in the taste perception and the reward processing have been identified in subjects with obesity [for review, see (6, 7)]. Consistent with this assumption, a reduction of the orosensory perception of fat stimuli associated with an overconsumption of lipid-rich foods was found in some patients with obesity (8).

Obesity surgery is an efficient method to induce a long-
term body weight loss and to correct obesity-associated
metabolic dysfunctions in subjects with a morbid obesity
(BMI ≥ 40 kg/m²) (9). Among the different bariatric procedures
developed, the vertical sleeve gastrectomy (VSG) consists to
remove two/third of the stomach. By combining efficient weight
and metabolic benefits with a low post-operative complication
rate, VSG is actually the most popular bariatric surgery
worldwide (10). Interestingly, most of the patients having
undergone a VSG report a change of their flavor perception
associated with a reduced interest for high-fat foods (11).
This healthier eating behavior suggests that VSG could reverse
the obesity-mediated decrease of the orosensory perception
of lipids, at least of some subjects. Since no systematic
relationship between the BMI loss and the improvement of
fat taste sensitivity is observed in humans (12), the underlying
mechanism remains elusive.

The oral cavity is one of the most abundant and
diversified microbial community within the body (13). The
greatest bacteria abundance is found upon the dorsal tongue
where microbial communities harbor a very complex spatial
pattern due to specific ecological niches (14). Interestingly, the
circumvallate papillae (CVP), housing most of the oral taste
buds, exhibit a dome-shape structure with a circular cleft which
could favor the development of specific bacterial communities

with the potential to affect the host cell functions. Consistent
with this assumption, an impairment to sense lipids was found
in obese subject displaying a specific CVP microbiota (15).

Collectively, these observations raise a basic question: is
there a relationship between the peri-papillae microbiome
and the orosensory lipid perception after VSG? To explore
this issue, oral microbiota samples in the direct vicinity
of CVP were analyzed and the lipid perception thresholds
were explored in volunteers with a severe obesity before and
6 months after surgery. Two complementary end-points have
been investigated: the impact of VSG on the CVP microbiota
composition and the relationship between CVP microbiota
and fat taste sensitivity using the 3-alternative force-choice
procedure (3-AFC) (16).

Subjects and methods

Subjects

The present clinical study is a part of the HumanFATaste2
program approved by French Ethic Committees (Comité de
Protection des Personnes, CPP n° 15-032 and Agence Nationale
de la Sécurité des Médicaments, ANSM n° 150811B-21)
and registered at Clinical Trials (NCT#02497274). Forty-four
patients with severe obesity (BMI ≥ 40 kg/m²) were included
in the HumanFATaste2 program (17). Among them, 32 patients
have undergone a VSG and were included in the present
work. Inclusion criteria were the following: women, aged 18
to 55 years, candidate for bariatric surgery based on criteria
established by international experts (BMI > 40 or >35 kg/m²
with severe comorbidities, after failure of multidisciplinary
care for a period of at least 1 year); and affiliation to a
health insurance. The exclusion criteria were the following:
taking medications known to alter taste, tooth decays, previous
surgical treatment of obesity, diabetes (hypoglycemic drug
intake or fasted plasma glucose level > 7 mmol/L), malignant
or chronic inflammatory diseases and tobacco use, alcohol or
drugs abuse. All subjects received detailed information about
the study and provided a written consent. To avoid gender-
mediated bias (18) and inter-individual variability, this study
was conducted on women only, each patient being her own
control (before and after VSG).

Vertical sleeve gastrectomy was performed laparoscopically, as previously described (19). Pre- and post-operative multidisciplinary managements were performed at the Louis Mourier Hospital (Assistance publique hôpitaux de Paris-APHP) according to French recommendations of the Haute Autorité de Santé (HAS) (20). Analysis of the body composition including fat mass using multifrequency impedancemetry (Seca, France), peri-papillae microbiota samples, blood collection and questionnaire on dietary habits (4-days food recall) were performed before and 6 months after VSG (Figure 1A). Plasma

<footnote>Abbreviations: 3-AFC, 3-alternative force-choice procedure; CVP, circumvallate papillae; KEGG, Kyoto encyclopedia of genes and genomes; LA, linoleic acid; LEfSe, Linear discriminant analysis effect size; LPS, lipopolysaccharides; VSG, vertical sleeve gastrectomy; OTU, operational taxonomic unit.</footnote>
C-reactive protein (CRP) levels, used as early biomarker of
inflammation, were assessed by using a ELISA kit (Enzo Life
sciences, France) in the same laboratory for all subjects. Six
months after VSG, patients have to assess their change in
gustatory and olfactory perception [taste and olfaction changes
(TOC) questionnaire (21)], adapted to include questions about
fat-mediated sensations. An analogic scale scored from 1 (no
change) to 10 (maximal change) was used to evaluate the
subjective gustatory and olfactory perception.

Oral microbiota samples and analysis

To delineate whether VSG is associated with changes in the
gustatory papillae microbiota ecology, swab samples before and
6 months after surgery were taken from each participant. Oral
microbiota samples were taken in the morning in overnight
fasted subjects directly from the V-shaped row of the CVP
located at the back of the tongue dorsa by smear (20 s) with
a sterile swab according to the previously published procedure
(15). Subjects were instructed not to brush their teeth or use
mouthwash in morning prior to oral swab in order not to
modify the oral flora. The bacterial composition before and after
surgery was determined by Vaiomer (Toulouse-Labège, France)
by sequencing variable regions of the 16S rRNA bacterial gene.
The V3-V4 hyper-variable regions of the 16S rRNA gene were
amplified from the DNA extracts during a first PCR step using
universal 16S primers V2 (Vaiomer, Toulouse-Labège, France).
The expected amplicon lengths were between 350 and 500 base
pairs (bp). For each sample, a sequencing library was generated
by addition of sequencing adapters and multiplexing indexes
during a second PCR step before sequencing on an Illumina
MiSeq machine in 2 × 300 bp paired-end reads mode as
described previously (22, 23).

The targeted metagenomic sequences from microbiota
were analyzed using the bioinformatics pipeline established
by Vaiomer to find operational taxonomic units (OTUs)
with Galaxy solution (FROGS) guidelines (24). Briefly, after
demultiplexing of barcoded Illumina paired reads, single-
read sequences were cleaned, the last 10 and 20 bases of
respectively, R1 and R2 reads were trimmed and paired into
longer fragments. OTUs were produced with single-linkage
clustering using two passes of the FROGS embedded swarm
algorithm: the first pass with an aggregation distance equal to 1
and the second pass with an aggregation distance equal to 3. The
taxonomic assignment was performed by BLAST against SILVA
132 database to determine bacterial profiles from phylum to
genus, and when reachable to species level. The following filters
were applied: amplicons with a length below 350 nt or a length
above 500 nt were removed and OTUs with abundance lower
than 0.005% were removed. Alpha and beta diversity analyses
were conducted on the OTU table.

The functional metagenome was inferred from the
clustered 16S sequences using the PICRUSt software (version
1.0.0) as per the instructions provided for the Genome
Prediction Tutorial for PICRUSt<fnref n="1" /> with recommended
scripts and default settings (25). As described in the
PICRUSt tutorial, the sequences previously grouped into
OTUs were processed through the QIIME closed reference
OTUs picking tool with a 97% similarity threshold to
obtain a set of OTUs IDs from the Greengenes reference
collection (gg_otus_13_5.tar.gz) as input for prediction
of corresponding metagenomes by PICRUSt. Through
this inference process, the abundance values of each
OTU were normalized to their respective predicted 16S
rRNA gene copy numbers and then multiplied by the
respective gene counts for metagenome prediction. PICRUSt
was also used to calculate the nearest sequenced taxon
index (NSTI) to quantify dissimilarity between reference
genomes and the predicted metagenomes. The resulting
core output was a list of Kyoto encyclopedia of genes and
genomes (KEGG) orthologues and predicted gene count
data for each sample. We used in house scripts to parse
the output into KEGG module categories for functional
pathways and structural complex hierarchies using the
KEGG database.<fnref n="2" /> The output matrix containing the relative
abundance of KEGG orthologous groups (KO) per sample
was processed with the online Galaxy interface for linear
discriminant analysis effect size (LEfSe) using an alpha
parameter significance threshold for the Kruskal-Wallis
test among classes set to 0.05 and the logarithmic linear
discriminant analysis (LDA) score cut-off was set to 2.0.
Respective cladograms were generated with modules at
the lowest level. Quantitative plots of differential features
were generated from normalized module level predicted
gene data showing means with standard deviation using
GraphPad Prism 6 software (GraphPad Software, La Jolla, CA,
USA).

Oral LA-threshold perception

The orosensory perception of lipids was determined
at 9 a.m. in fasting patients using the 3-AFC method
with the linoleic acid (LA) as lipid model (15). LA was
chosen because this polyunsaturated long-chain fatty
acid, widely found in foods, binds with a high affinity
the lipid receptors expressed in the taste buds (i.e., CD36
and GPR120) (26). Patients checked (sip and spit) sets
of three samples (two with the control solution, one
with LA in the control solution), and had to identify the

<footnote n="1">1 http://picrust.github.io/picrust/tutorials/genome宪 prediction.html#
genome-prediction-tutorial</footnote>

<footnote n="2">2 http://www.genome.jp/kegg/module.html</footnote>
sample that was different from the two others. Sets were presented in an ascending concentration (from 0.00028 to 5% LA) spaced by 0.25 log units (18 solutions in total). The procedure was stopped when the LA sample was correctly identified three times consecutively. This LA concentration represented the perception threshold value of the patient.

The protocol used to prepare LA solution is fully detailed elsewhere (8, 16). In brief, LA (Sigma Aldrich, France) oil-in water emulsions were prepared in a solution of 5% acacia gum (wt/wt; Fluka, USA), 5% mineral oil (wt/wt; Cooper, France), and 0.01% EDTA (wt/wt; VWR international, USA) diluted in mineral water. Acacia gum and paraffin oil were added to limit viscosity and lubricity differences between control and experimental samples. EDTA was added to prevent the oxidation of LA (16). Samples were mixed conventionally by using a stirrer (Corning, France) and homogenized with a sonicator (Sonics Materials Inc., USA). The duration of sonication was adapted to LA concentrations to obtain a similar particle size and, thus minimize textural influences (8). In all cases, sonication was conducted by lapses of 30 s separated by a 1 min pause. Sonication was conducted in a hermetic chamber saturated with nitrogen and beakers were cooled by using an ice bath to limit the formation of oxidized compounds during emulsion preparation.

Samples were presented as 5 ml portions in opaque cups and were tested at room temperature. Subjects were instructed to hold the 5 ml solution in their mouth for 7 s, spit the solution out, and wait for 20 s before tasting the next sample. The interval between 2 sets was 60–120 s, during which participants were asked to rinse their mouths with water. Testing was conducted under red lighting and with participants wearing a nose clip to limit visual and olfactory inputs, respectively. To reduce the potential nutritional interferences on the taste perception due to the last meal before the 3-AFC sessions, all subjects were asked to eat for the dinner, the following standardized meal: raw tomato (100 g) + 1 teaspoon of sunflower oil (5 g), bread (20 g of French baguette), chicken breast (120 g), potatoes cooked in water (250 g), green beans (100 g), butter (5 g), 1 yoghurt sweetened with cane sugar, 1 standard apple compote. All patients indicated that they complied with this protocol in its entirety.

## Statistical analysis

**Microbiome analysis:** Significant variations in alpha diversity were assessed using the Kruskal-Wallis test or the Wilcoxon rank-sum test. Multidimensional scaling analyses were performed on beta diversity distance matrices and

differences between groups were assessed using PERMANOVA and PERMDISP analyses (2,000 permutations). LEfSe analyses were used to determine significant differences in taxa relative abundance between groups (27) using default parameters (alpha parameter significance threshold set to 0.05 and the logarithmic LDA score cutoff set to 2.0).

Other analyses: After a normality test (D'agostino and Pearson), data were analyzed with either an unpaired Mann-Whitney test or a paired Wilcoxon test for paired analysis. Unpaired Mann-Whitney, Wilcoxon tests, and Linear correlation tests were performed using GraphPad Prism (GraphPad Software, USA). Data from 4-days food recall and TOC questionnaires were compared using Student's test (for means) and Chi-square or Fischer's exact test (for percentages).

## Results

### Post-VSG changes in body composition and modifications of tongue microbiota surrounding the circumvallate papillae

Mean of BMI and age of the study population were 43.1 ± 0.7 kg/m² and 38.4 ± 1.3 years, respectively. The time-course of the study including the main anthropometric parameters changes are shown in **Figure 1A**. Six months after VSG, patients displayed a drastic reduction of body weight, BMI, and fat mass associated with a 2-fold decrease of plasma CRP levels (**Figure 1B**). Analysis of taxonomic diversity at the genus level has pointed out a reduction of CVP microbial richness after VSG (Shannon's index, **Figure 1C**) with a composition clearly distinct from that observed before surgery (Bray-Curtis beta-diversity, **Figure 1D**) as shown by the PERMANOVA test (*p*-value = 0.0005). To identify the differential taxa abundance, a LEfSe analysis with the 16S rRNA gene sequence data was performed (**Figure 1E**).

At the genus level, an enrichment of CVP microbiome in *Prevotella*-6, *Leptotrichia*, *Actinomyces*, and *Campylobacter* was found after VSG whereas abundance of *Porphyromonas*, *Fusobacterium*, *Neisseria*, *Prevotella*-2, *Parvimonas*, and *Pseudomonas* was reduced. Several differential pathways and structural features in the predicted metagenomes were identified from the PICRUSt analysis. LEfSe cladograms of KEGG pathways and structural components derived from pairwise group analysis of pre- and post-VSG patients are shown in **Figure 2**.

After VSG, the predicted metagenomic profile of microbiota surrounding CVP was notably characterized by a rise of carbohydrate and glycoaminoglycan metabolism along with a decrease in lipid, lipopolysaccharides (LPS) and nitrogen metabolism (Figure 2).
<figure><img src="image_4.png" /></figure>

## Post-VSG improvement of LA perception threshold is associated with a specific peripapillae microbiome composition

The comparison of LA perception thresholds before and after VSG has allowed distinction of two subgroups: patients displaying a post-operative improvement of the fat taste sensitivity (i.e., a lower LA threshold) and unimproved patients (Figure 3A). This difference of oral lipid sensing was found to be independent of the body weight and fat mass loss after surgery, but was associated in the LA-improved group with a significant

reduction of plasma CRP levels, used as a marker of the systemic inflammatory status (28; Figure 3B).

To explore the relationship between peripapillae microbiota composition and orosensory perception of LA, bacterial diversity and taxonomy were compared in improved and unimproved patients. In contrast to what was found before surgery (Figure 3C), a statistically significant difference in alpha diversity discriminated LA improved from unimproved patients 6 months after VSG, bacterial richness at the genus level being reduced in LA improved subjects (Figure 3D). Similarly, whereas the taxonomic LEfSe analysis failed to reveal any difference between patient subgroups before VSG (Figure 3E), LA improved patients were characterized
<figure><img src="image_5.png" /><figcaption>FIGURE 2</figcaption></figure>

a: Central carbohydrate metabolism

b: Glycoaminoglycan metabolism

c: Lipid metabolism

d: Lipopolysaccharide metabolism

e: Nitrogen metabolism

f: Co-factor and vitamin biosynthesis

g: Cys and Met metabolism

h: Lys metabolism

i: Other amino acid metabolism

j: Biosynthesis of secondary metabolites

Predicted metagenomics LEfSe changes induced by VSG (before vs. after surgery). Cladogram showing the KEGG metabolic pathway hierarchy represented by rings with the consolidated pathway modules at the outmost ring. KEGG modules are shaded in color according to the group in which it is most abundant. Histogram showing the relative predicted counts for each differential feature with corresponding letters in the cladogram. Numbers at the center of the cladogram correspond to major pathway groupings: 1- Carbohydrate and lipid metabolism; 2- Energy metabolism; 3- Nucleotide and amino acid metabolism; 4- Secondary metabolism. VSG, vertical sleeve gastrectomy.

by a greater abundance of *Prevotella* 7 and *Atopobium* associated with a reduction of *Porphyromonas*, *Fusobacterium*, and *Haemophilus* bacteria (Figure 3F). To gain insight into relationships between these taxonomic differences and LA perception thresholds, linear regression analyses were conducted after surgery. As shown in Figure 4A, significant negative correlations were found between LA perception thresholds and *Atopobium* (*r* = 0.40, *p* = 0.02) and *Prevotella* 7 (*r* = 0.36, *p* = 0.04), a high bacteria level being associated with a low threshold and, thus, a high lipid sensitivity. An opposite scenario was found in patients with *Porphyromonas* (*r* = 0.36, *p* = 0.04–Figure 4A). The multivariate analysis (principal component analysis–PCA) revealed the existence of a strong relationship between the studied variables, the values on the x- and y-axes, describing the component score of the dimensions 1 and 2, accounting for 57.7 and 18.8% of inertia respectively, (total 76.5%, Figure 4B). Confidence ellipse analysis suggested that the oral microbiota composition in the direct vicinity of CVP was sufficient to distinguish LA-improved from unimproved patients. In line with this assumption, the metagenomic analysis highlighted that the peri-papillae microbiota from LA-improved patients was predominantly characterized by a high carbohydrate metabolic activity while it was mainly oriented towards lipids in unimproved patients (Figure 4C).

Six months after surgery, the daily energy intake was reduced in a same proportion in the two sub-groups (–55.1 and 52.0% in LA-improved and LA-unimproved patients, respectively), the daily carbohydrate, lipid and protein intakes being overall similar (Table 1). Whether a comparable change of sweet, salty, and bitter taste intensity and olfactory performance was reported by the two groups of patients, LA-improved patients displayed a more marked upward trend for the fat taste intensity change than unimproved ones (*p* = 0.04–Table 2).

Discussion

A body of evidence supports the existence of a sixth taste modality devoted to the perception of dietary lipids (26, 29–31). Interestingly, studies report that the taste of fat can be compromised in obesity (7, 31). Although this disturbance may be associated with a preferential consumption of fat-rich foods (8), underlying mechanisms leading to this obesogenic behavior remain poorly understood. As found at the gut level, an oral microbiota dysbiosis is usually observed in subjects with obesity (32). Moreover, an association between the bacterial composition surrounding gustatory papillae and fat taste sensitivity was recently reported in some obese subjects irrespectively from BMI changes (15, 33, 34). The present study strengthens this finding and provides new insights
<figure><img src="image_6.png" /><figcaption>FIGURE 3</figcaption></figure>

Vertical sleeve gastrectomy (VSG)-associated changes in the orosensory perception of a lipid model (linoleic acid, LA) and in the composition of tongue coating microbiota surrounding the circumvallate gustatory papillae. (A) LA perception thresholds before and 6 months after VSG (Mann-Whitney test). (B) Comparison of body composition and plasma C-reactive protein (CRP) levels in LA improved and LA not improved patients (Mann-Whitney test). (C,D) Comparison of peri-papillae microbiota diversity (Shannon index at the genus level) before and after VSG in LA improved and LA not improved patients. (E,F) Taxonomic differential LEfSe analysis before and after VSG on peri-papillae microbiota in LA improved and LA not improved patients. Cladogram of taxonomic assignments from 16S rRNA gene sequence data. Taxa are shaded according to the group in which it is most abundant. Histogram showing the percent relative abundance with corresponding letters in the cladogram. Means ± SEM. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001.

on relationships between obesity surgery and orosensory perception of lipids.

Six months after VSG, the taxonomic diversity at the genus level was found to be reduced in direct vicinity of CVP in comparison to the pre-operative period. This surgery-associated change counteracted the greater alpha diversity currently found in oral microbiota from subjects with obesity (35, 36). Analysis of beta-diversity before and after VSG also revealed a compositional dissimilarity of peri-papillae microbiota communities. Consistent with this data, taxonomic analysis has identified seven bacterial genera, of which the relative abundance was statistically different before and after

VSG. By changing the gut microbiota composition, it was previously shown that VSG contributes to the improvement of host metabolism impaired during obesity (37–40). We show herein that VSG is also associated with a remodeling of the tongue microbiome in the direct vicinity of CVP, an event that might affect the host gustatory functions.

Consistent with this assumption, the present study also shows that changes in the orosensory perception of lipids coincide with post-VSG changes in the CVP microbiome. Indeed, CVP microbiota composition was found to be dissimilar between patients with and without improvement of their LA perception thresholds after VSG. Five bacterial genera
<figure><img src="image_7.png" /></figure>

of which the relative abundance was statistically different
were identified. Among these, the level of *Prevotella* genus,
considered as a marker of the fiber-rich foods intake (41, 42),
was higher in improved patients. Bacteria from *Fusobacterium*
and *Porphyromonas* genera also segregate these two sub-groups,
their abundance being higher in LA-unimproved patients
suggesting a pro-inflammatory signature. Indeed, cytokines
produced by these bacteria are known to promote a local
inflammation (43). A pro-inflammatory environment in the
direct vicinity of CVP might have a deleterious effect on the fat
taste perception by reducing taste bud density and turnover (44),
an event known to specifically reduce the fatty taste sensitivity in
humans (45). The fact that LA-unimproved patients reported a

lower perceived fat intensity than improved patients (Table 2)
is consistent with this hypothesis. According to a previous
observation done in obese volunteers from another cohort
(15, 34), we have found that *Porphyromonas* abundance and LA
perception threshold tends to be positively associated after VSG
(i.e., high bacteria level = high LA threshold and thus lower LA
perception sensitivity). Collectively, these data strongly suggest
that VSG might affect the oral perception of lipids by modifying
lingual microbiota in some patients.

Although correlation is not causation, it was tempting to
speculate that this new microbial balance in direct vicinity
of gustatory papillae might participate to the healthier food
choice reported by some patients having undergone a VSG (46).
<table><caption>TABLE 1 Reported food intake (4-days food recall) before (pre-op) and 6 months (post-op) after surgery.</caption><thead><tr><th>Variables</th><th>LA-improved (n=14)</th><th>LA-unimproved (n=18)</th><th>LA-I vs. LA-U (post-op)</th></tr></thead><tbody><tr><td><strong>Food intake (g/d)</strong></td><td></td><td></td><td></td></tr><tr><td>Pre-op</td><td>1145.6±156</td><td>1193.6±271</td><td>ns</td></tr><tr><td>Post-op</td><td>570.1±184</td><td>567.5±219</td><td></td></tr><tr><td><strong>Energy intake (Kcal/d)</strong></td><td></td><td></td><td></td></tr><tr><td>Pre-op</td><td>1589.1±237</td><td>1545.4±308</td><td>ns</td></tr><tr><td>Post-op</td><td>875.5±314</td><td>804.3±191</td><td></td></tr><tr><td><strong>Carbohydrates (% energy intake)</strong></td><td></td><td></td><td></td></tr><tr><td>Pre-op</td><td>45.4±5.3</td><td>46.3±6.4</td><td>ns</td></tr><tr><td>Post-op</td><td>44.2±9.7</td><td>43.6±7.3</td><td></td></tr><tr><td><strong>Lipids (% energy intake)</strong></td><td></td><td></td><td></td></tr><tr><td>Pre-op</td><td>34.8±4.7</td><td>33.3±5.6</td><td>ns</td></tr><tr><td>Post-op</td><td>36.6±9.1</td><td>37.9±6.1</td><td></td></tr><tr><td><strong>Proteins (% energy intake)</strong></td><td></td><td></td><td></td></tr><tr><td>Pre-op</td><td>19.8±2.2</td><td>20.3±4.0</td><td>ns</td></tr><tr><td>Post-op</td><td>19.2±3.1</td><td>19.7±4.9</td><td></td></tr></tbody></table>

Means ± SD; ***p<0.001; ns, not significant. Pre-op vs. post-op, paired student. I vs. NI, student. Op, operated.

Analysis of 4-days food recall questionnaires invalidate this hypothesis. Indeed, similar daily caloric intake and energetic nutrient distribution being found in two sub-groups of patients.

<table><caption>TABLE 2 Reported gustatory and olfactory changes [taste and olfaction changes (TOC) questionnaire] 6 months after surgery.</caption><thead><tr><th>VSG</th><th>LA-improved (n=14)</th><th>LA-unimproved (n=18)</th><th>p</th></tr></thead><tbody><tr><td><strong>Taste changes</strong></td><td></td><td></td><td></td></tr><tr><td>Increased global taste (% of patients)</td><td>64.3</td><td>50</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>6.6 ± 1.9</td><td>6.2 ± 2.2</td><td>ns</td></tr><tr><td>Increased sweet taste (% of patients)</td><td>71.4</td><td>83.3</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>6.1 ± 2.8</td><td>4.1 ± 3.1</td><td>ns</td></tr><tr><td>Increased fat taste (% of patients)</td><td>71.4</td><td>66.7</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>7.7 ± 1.8</td><td>5.5 ± 2.9</td><td>0.04</td></tr><tr><td>Increased salt taste (% of patients)</td><td>64.3</td><td>38.9</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>6.3 ± 1.7</td><td>4.6 ± 2.9</td><td>ns</td></tr><tr><td>Increased bitter taste (% of patients)</td><td>7.1</td><td>16.7</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>6.0 ± 0.0</td><td>6.0 ± 2.0</td><td>ns</td></tr><tr><td><strong>Olfactory change</strong></td><td></td><td></td><td></td></tr><tr><td>Increased olfaction (% of patients)</td><td>35.7</td><td>44.4</td><td>ns</td></tr><tr><td>Intensity of change (1-10)</td><td>6.4 ± 1.7</td><td>5.3 ± 1.8</td><td>ns</td></tr></tbody></table>

Means ± SD; ns, not significant; p = X² test of Fisher exact test.

Such a disconnection between subjective food sensations and measured oral fat perception has already been found in human and seems to be dependent from methods of investigation used [for a review, (47)].

It is noteworthy that no systematic improvement of the orosensory lipid sensitivity was found after VSG irrespectively of body weight and fat mass loss (Figure 3B). This interindividual variability was already reported in obese subjects (15, 34). Interestingly, a pro-inflammatory bacterial signature in direct vicinity of CVP characterized the obese non-lipid tasters (15). The present study confirms this previous observation and strengthens the hypothesis of an oral microbiota contribution to inter-individual variability of oral lipid detection in human with obesity. However, this phenomenon is likely multiparametric. In previous studies conducted with the same cohort, the plasma Kynurenine levels and the LA perception threshold were found to be positively associated only in the LA- improved group suggesting that Tryptophan/Kynurenine metabolism is one of regulatory factors in the cross-talk between nutritional obesity, bariatric surgery, and fat taste (48). By contrast, despite digestive tract modifications secondary to surgery, any correlation was found between the fat taste sensitivity and the VSG-mediated modifications of main appetite-regulating hormones from the digestive tract, i.e., ghrelin, insulin, GLP-1, and PYY (17).

The main weakness of this study is the low number of patients in each sub-groups (improved, n = 14 vs. unimproved, n = 18). Nevertheless, investigations being performed only in selected women who were their own controls (before vs. after VSG) has likely reduced the possible biases due to gender, interindividual variability et taste alterations. Despite this limitation, LA-improved and not improved patients were clearly distinguished after VSG contrary to what is observed before surgery. Another limitation is the use of 4-days food recall to evaluate the eating habits of patients after surgery. Although the
self-reporting methods easy to implement are commonly used, they may generate biased responses (47). Studies using a direct method to accurately measure the changes in food selection (e.g., *ad libitum* buffet paradigm (49)) will be required to confirm the present data.

In brief, this study shows that VSG is associated with changes in the microbiota composition surrounding the gustatory papillae, this event being coinciding with an improvement of the orosensory perception of lipids in some patients, irrespectively from their BMI. This original observation raises at least two basic questions for future investigations: why this VSG-induced fat taste improvement is not systematically reproducible in human? Does it constitute a long-term health advantage after obesity surgery? Corroboration of present data with larger selection of obese subjects might lead to the validation of easily assayed predictive bacterial biomarkers useful to identify the patients with improved orosensory lipid perception from unimproved patients in order to provide them personalized nutritional advices ensuring the long-term success of the VSG.

## Data availability statement

The data presented in the study are deposited in the ENA repository, accession number PRJEB58719.

## Ethics statement

The studies involving human participants were reviewed and approved by Comité de Protection des Personnes, CPP n° 15-032 and Agence Nationale de la Sécurité des Médicaments, ANSM n° 150811B-21) and registered at Clinical Trials (NCT#02497274). The patients/participants provided their written informed consent to participate in this study.

## Author contributions

PB, SL, and XC: conceptualization. LR, AB, FS, and JC: methodology. PB: formal analysis, writing – original draft

preparation, supervision, and funding acquisition. PB, AB, SL, XC, FS, JC, and VB-B: writing – review and editing. AB: project administration. All authors contributed to the article and approved the submitted version.

## Funding

The HumanFATaste2 program from which this study is derived is supported by a grant from the French National Research Agency under the program "Investissements d'avenir" (ANR-11-LABX-0021-LipSTIC) and by financial supports from the Lesieur/Avril group, Tereos company, and Benjamin Delessert Institute.

## Acknowledgments

We thank Pascaline Delaby (Lesieur company) and Cindy Le Bourgot (Tereos company) members of the HumanFATaste2 consortium for fruitful discussions. We are grateful to Vincent Azalbert (UMR 1297 INSERM/Univ Toulouse 2) for management of oral microbiota samples before sequencing.

## Conflict of interest

FS is employed by Vaiomer.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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