The Associations between Biochemical and Microbiological … · 2021. 1. 14. · ResearchArticle The Associations between Biochemical and Microbiological Variables and Taste Differ

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The Associations between Biochemical andMicrobiological Variables and Taste Differ in Whole

Saliva and in the Film Lining the TongueYunzi Feng Heacutelegravene Licandro Christophe Martin Chantal Septier Mouming

Zhao Eric Neyraud Martine Morzel

To cite this versionYunzi Feng Heacutelegravene Licandro Christophe Martin Chantal Septier Mouming Zhao et al The Asso-ciations between Biochemical and Microbiological Variables and Taste Differ in Whole Saliva and inthe Film Lining the Tongue BioMed Research International Hindawi Publishing Corporation 20182018 pp1 - 10 10115520182838052 hal-01907780

Research ArticleThe Associations between Biochemical and MicrobiologicalVariables and Taste Differ in Whole Saliva and in the FilmLining the Tongue

Yunzi Feng12 Heacutelegravene Licandro3 Christophe Martin2 Chantal Septier2 Mouming Zhao 1

Eric Neyraud 2 andMartine Morzel2

1School of Food Science and Engineering South China University of Technology Guangzhou 510640 China2Centre des Sciences du Gout et de lAlimentation AgroSup Dijon CNRS INRA Universite de Bourgogne Franche-Comte21000 Dijon France3UMR A 02102 Procedes Alimentaires et Microbiologiques (PAM) AgroSup Dijon Universite de Bourgogne Franche-Comte21000 Dijon France

Correspondence should be addressed to Eric Neyraud ericneyraudinrafr

Received 10 January 2018 Accepted 21 May 2018 Published 14 June 2018

Academic Editor Asta Tvarijonaviciute

Copyright copy 2018 Yunzi Feng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this work was to investigate whether the biological film lining the tongue may play a role in taste perception Forthat purpose the tongue film and saliva of 21 healthy subjects were characterized focusing on microorganisms and their mainmetabolic substrates and products In parallel taste sensitivity was evaluated using a test recently developed by our group and thelinks between biological and sensory data were explored by a correlative approach Saliva and tongue film differed significantly inbiochemical composition (proportions of glucose fructose sucrose and lactic butyric and acetic acids) and in microbiologicalprofiles compared to saliva tongue film was characterized by significantly lower proportions of Bacteroidetes (plt0001) and itsmain genus Prevotella (plt001) and significantly higher proportions of Firmicutes (plt001) Actinobacteria (plt0001) and thegenus Streptococcus (plt005) Generic taste sensitivity was linked to biological variables in the two compartments but variablesthat appeared influent in saliva (flow organic acids proportion of Actinobacteria and Firmicutes) and in tongue film (sugars andproportions of Bacteroidetes) were not the same This study points to two interesting areas in taste research the oral microbiomeand the specific characterization of the film lining the tongue

1 Introduction

Eating behavior is a key factor of health in humans someof the major pathologies affecting the modern societies suchas obesity cardiovascular diseases or type 2 diabetes havebeen reported to be strongly linked to it Determinantsof eating behavior are various and comprise for examplebiological psychological and socioeconomic factors Amongbiological factors the sense of taste participates in thesensory perception of the food and thus influences foodchoices Impairments in taste perception can for examplelead to eating disorders and malnutrition [1] Probably themost objective way of characterizing and classifying subjectsaccording to their taste function is to measure their taste

sensitivity and more precisely their individual detectionthreshold This approach is thus widely used in patients withoral complaints [2 3] or with diet-related conditions such asobesity [4 5]

Taste perception occurs after activation of specializedreceptors in the taste buds on the tongue Saliva is theprincipal fluid that interacts with food and it is the mediumthat bathes the taste buds thus it plays an important role intaste perception through several mechanisms such as protec-tion of the taste receptors or transport of taste compounds[6] Several studies have indeed described the relationshipbetween salivary composition and taste sensitivity for exam-ple to bitterness [7ndash9] or to the taste of oleic acid [10] Besidesfree-flowing saliva the biological film lining the tongue

HindawiBioMed Research InternationalVolume 2018 Article ID 2838052 10 pageshttpsdoiorg10115520182838052

2 BioMed Research International

Table 1 Primers used for qPCR assays

Primer Oligonucleotide sequence (51015840-31015840) Target ReferenceSPU Fwd AAACTCAAAKGAATTGACGG All bacteria aSPU Rev CTCACRRCACGAGCTGAC All bacteria aACT Fwd TACGGCCGCAAGGCTA Actinobacteria aACT Rev TCRTCCCCACCTTCCTCCG Actinobacteria aBACT Fwd CRAACAGGATTAGATACCCT Bacteroidetes aBACT Rev GGTAAGGTTCCTCGCGTAT Bacteroidetes aFIRM Fwd TGAAACTYAAAGGAATTGACG Firmicutes aFIRM Rev ACCATGCACCACCTGTC Firmicutes a120574-P Fwd TCGTCAGCTCGTGTYGTGA 120574-Proteobacteria a120574-P Rev CGTAAGGGCCATGATG 120574-Proteobacteria a120573-P Fwd ACTCCTACGGGAGGCAGCAG 120573-Proteobacteria b120573-P Rev TCACTGCTACACGYG 120573-Proteobacteria bFuso Fwd CGCAGAAGGTGAAAGTCCTGTAT Fusobacterium spp cFuso Rev TGGTCCTCACTGATTCACACAGA Fusobacterium spp cVeil Fwd A(CT)CAACCTGCCCTTCAGA Veillonella spp dVeil Rev CGTCCCGATTAACAGAGCTT Veillonella spp dStrep Fwd GTACAGTTGCTTCAGGACGTATC Streptococcus spp eStrep Rev ACGTTCGATTTCATCACGTTG Streptococcus spp ePrev Fwd CACCAAGGCGACGATCA Prevotella spp fPrev Rev GGATAACGCCYGGACCT Prevotella spp fa De Gregoris et al 2011 b Pecastaings et al 2016 c Suzuki et al 2004 d Rinttila et al 2004 e Picard et al 2004 f Marathe et al 2012

surface is even more intimately in contact with the tastebudsThenumerous depressions of the tongue dorsum formaunique ecological site which provides a large surface area forthe accumulation of saliva oral debris and microorganisms[11] Thus the tongue is coated with a film comprisingbacteria desquamating cells and residual saliva The termldquotongue coatingrdquo is also used in the literature althoughit very often refers to an undesirable excess of biologicalmaterial Some studies have linked tongue coating to tasteperception Lower overall taste performance (sensitivity tofour tastants) was marginally observed in elderly patientswith a coated tongue [3] and reduced recognition thresholdsof saltiness and acid were observed in nursed elderly afterlight scraping the anterior half of the tongue [12] Both casessupport the assumption that the film lining the tongue mayinfluence taste perception and we formulate the hypothesisthat more particularly the bacterial component of this filmdeserves attention This is based on several direct or indirectobservations suggesting that oral bacteria could modulatethe sense of taste Thus Solemdal et al [13] have made theconnection between higher salivary counts of the bacteriaStreptococci and Lactobacilli and reduced perception of sourtaste We also observed that a low sensitivity to the tasteof fatty acids was associated with a high concentration oforganic acids probably of bacterial origin in saliva [10] Twopossible mechanisms were mentioned first higher bacterialloads in the tongue filmwould set a barrier limiting the accessof taste molecules to the taste receptors second bacterialmetabolism may modulate the concentration of tastants(eg substrates such as sugars or amino-acids end-productssuch as organic acids) near the taste receptors and thus

modify taste sensitivity according to the sensorial adaptationmechanism

In this context this study had two main objectives firstit aimed to characterize the composition of saliva and tonguefilm in healthy subjects focusing on microorganisms andtheir main metabolic substrates and products (sugars andorganics acids) Second it aimed to investigate whethervariability in these indices especially microorganism profilewas related to variability in sensitivity to the five basic tastes(sweet sour salty bitter and umami)

2 Materials and Methods

21 Chemicals and Reagents Fructose lactose sucrose aceticacid propionic acid butyric acid and lactic acid wereobtained commercially from Sigma-Aldrich (Steinheim Ger-many) Glucose was purchased from Merck (DarmstadtGermany) The primers (Eurogentec Belgium) used forpreamplification reaction (PCR) are shown in Table 1

22 Subjects and Sampling of Saliva and Tongue Film Thestudy and protocols were approved by a relevant ethical com-mittee (Comite de Protection des PersonnesOuest V n∘ 2016-A01954-47) Written informed consent was obtained fromthe participants Twenty-one healthy subjects (11 females 10males 22 to 60 years old) who all declared themselves to bein good oral health participated in this study More preciselythe exclusion criteria were as follows smokers pregnantwomen food allergy sufferers long term (over one month)medicated subjects subjects who took an antibiotic coursehad dental treatment or used an antiseptic mouthwash in thepreceding month subjects who ever received head and neck

BioMed Research International 3

radiotherapy and sufferers from pathologies affecting theoral cavity (eg Sjogren syndrome lichen planus gingivitis)In addition none of the subjects brushed their tongue aspart of their oral hygiene routine Donors were instructednot to eat or drink at least 2h before sample collectionwhich occurred between 10 and 11 am Unstimulated wholesaliva was collected by direct draining into a 5 ml weighedtube during 3 minutes After a short rest seated participantsswallowed residual saliva immediately stuck their tongueout as far as possible and maintained this pose while thefilm was taken All samples were collected by the sameexperimenter Tongue film was collected by scraping thetongue with a plastic sterile stick from the root to the apexapplying one single scraping motion The whole samplingprocedure was applied on two separate days per subjectOn the first day weights were recorded and samples wereprepared for biochemical and microbiological analyses Onthe second day the pH of saliva and film samples wasmeasured immediately after collection with a microelectrode(IQ240 pH meter IQ Scientific Instruments Carlsbad CAUSA)

23 Determination of Sugars and Acids Concentration byHPLC Saliva was diluted 12 and tongue film 14 with MilliQ water Samples were centrifuged at 15000 g for 15 min at4∘C and the supernatant was further diluted 110 and filteredthrough a 022 120583m nylon filter

Sugars were analyzed using Dionex ICS-3000 ion chro-matographic system (Dionex Sunnyvale USA) consistingof a gradient pump chromatography enclosure with a 5-120583L injection loop and an electrochemical detector Thesugar composition of the sample was determined by pulsedamperometric detector (PAD) using a CarboPac 1 column (2times 250 mm id 5 mm Dionex) and 100mM NaOH as mobilephase at a flow rate of 025 mLmin

Organic acids were also analyzed byDionex ICS-3000 ionchromatography system (Dionex Sunnyvale USA) equippedwith an electroconductivity detector Ion chromatographywas carried out using IonPac AS11-HC column (Dionex4times250 mm) The mobile phase 08 mMNaOH was at a flowrate of 025 mLmin and at room temperature The organicacids were detected by chemical suppressed conductivityusing an anion-ICE micromembrane suppressor

Sugars and organic acids were identified and quantifiedaccording to the retention time and signal intensity ofreference compounds The standard curves were obtainedusing glucose fructose sucrose and lactose at concentrationsranging from 0005 to 25 mgL and lactate acetate propi-onate and butyrate at concentrations ranging from015 to 375mgL

24 Enumeration of Colony-Forming Units (CFU) Serialdilutions (10minus3 to 10minus6) of diluted saliva and film sampleswere prepared and 100 120583l samples were plated on Columbiamedium (Biokar) supplemented with glucose (10 gl) and5 (vv) defibrinated sheep blood Counting of colonieswas performed after incubating the plates in two differentconditions aerobic and anaerobic (5 CO

2) at 37∘C for 48

h The total concentration of cultivable microorganisms was

approximated by adding the concentrations of aerobic andanaerobic microorganisms

25 DNA Extraction and qPCR DNA extraction was per-formed using Nucleo-Spin DNA tissue kit (Macherey-NagelGermany) following the manufacturerrsquos instructions with aminor modification namely in the lysis step 01 120583m glassbeads were added and themixture was vortexed at maximumspeed for 10 min Quality of the extracted DNA was deter-mined using a NanoDrop ND-1000 spectrophotometer andgel electrophoresis PCR amplifications were performed intriplicate in 20 120583l reaction mixtures containing 4 120583l of DNAextract 300 nM of each primer and 10 120583l of SsoAdvancedUniversal SYBR Green Supermix (Bio-Rad) in a CFX96Real-Time PCR Detection System (Bio-Rad USA) Thermalcycling conditions were an initial denaturation at 95∘C for 3min followed by 40 cycles of 95∘C for 15 s and 60∘C for 30 sEfficiency of amplificationwas determined by running a stan-dard curve for each primers couple Primers were selectedto target total bacteria and the most representative phylaclasses or genera of oral bacteria (Table 1) All primers tar-geted 16S rRNA except for Streptococcus spp whose primerstargeted tuf gene The percentage (P) of each specific group(SPE) was determined relatively to the total bacteria (SPU)with the equation P=(Eff SPU)Ctspu(EffSPE)Ctspex100 withEff being the PCR amplification efficiency Preliminary exper-iments revealed that 120572-Proteobacteria was not detected in theoral microbiota Results for the phylum Proteobacteria weretherefore calculated by adding results of the two classes 120573-Proteobacteria and 120574-Proteobacteria Similarly results of thegenus Fusobacteria were considered as an approximation forthe phylum Fusobacteria because it is almost exclusively theonly genus of this phylum found in the oral cavity [14]

26 Determination of Taste Sensitivity Scores Taste sensitivitywas evaluated for the five basic tastes sweet (fructose)salty (sodium chloride) sour (citric acid) bitter (quininehydrochloride) and umami (monosodium glutamate) Alltastants were of food grade quality and deionized water wasused as solvent For each taste the lowest perceived con-centrations were determined thanks to a test (Tsty test)recently developed by our group (Patent WO2015165880)This test uses test-sheets made from edible wafer paper Atest-sheet consists of six series of three precut discs (18 mmdiameter) For each series one disc contains the tastant (theldquotasty discrdquo) and the two others are neutral On one test-sheetthe tastant concentration increases gradually from the firstseries to the sixth series (Figure 1) The concentrations werechosen to obtain a Gaussian distribution of the individualscores across the general population

For each series subjects were instructed to taste thethree discs by placing them on the tip of the tongue fora few seconds and to find the tasty disc by answering thefollowing question Which disc is different from the othertwo Subjects were instructed to randomly answer if no dif-ference was perceived Answers recorded for a full test-sheetwere converted to a score ranging from 0 (low sensitivityhighest concentration not perceived) to 6 (high sensitivityall concentrations perceived) Calculation of the score was


Table 2 Salivary flow rates and weight of tongue film sampled (n=21)

Mean Min Max MedianStandard

SD deviation(SD)

Salivary flow rates 045 005 113 036 031 686(gmin)Weight of tongue film 0025 0007 0051 0020 0012 485(g)

Figure 1 Example of a test-sheet Tasty discs are indicated incolor with increasing color intensity corresponding to increasingconcentrations of the tastant Within each series of three discssubjects are asked to identify which one is the tasty disc

inspired by the Best-EstimateThreshold (BET) method eachtest-sheet resulted in a series of 6 answers (correctwrong)ordered by increasing concentration of tastant The scorecorresponds to the number of consecutive correct answersafter the highest concentration for which a wrong answer wasgiven For example a score of 2 corresponds to the case whereanswers given for the sixth and fifth series are correct butanswer for fourth series is wrong whatever the answers givenfor the other series For each taste the sensitivity scorewas theaverage calculated on four replicates

27 Statistical Analysis Thestatistical analysis was conductedusing Statistica (StatSoft) Paired t-tests were performed toevaluate the difference in pH and composition between salivaand tongue film Pearson correlation was calculated betweenpH in saliva and pH in film The correlation between tastesensitivity scores and biological data was evaluated usingSpearman correlation tests The choice of a nonparametrictest in that case is justified by the presence of censored data(scores = 0 or 6) in the sensory dataset A score of 0 indicates

that the subject would perceive the tastant only above thehighest concentration presented while a score of 6 indicatesthat the subject could perceive the tastant even below thelowest concentration presented

3 Results

31 pH of Saliva and Tongue Film The pH of saliva variedfrom 589 to 700 (mean 647 plusmn 029) and it was significantlylower (p lt 0001) than the pH of tongue film which variedfrom 653 to 786 (mean 715 plusmn 036) Moreover a significantcorrelation (r = 0839 p lt 0001) was observed between thepH of saliva and pH of film

32 Salivary FlowRate andWeight of Tongue Film Results aredepicted in Table 2 Saliva flow rates showed large variationsranging from 005 to 113 gmin (mean 045plusmn 031 gmin)Thewet weights of tongue film varied between 7 and 51 mg (mean25 plusmn 12 mg)

33 Sugars and Organic Acids Profiles The concentrationsand percentages of four sugars (glucose fructose sucroseand lactose) and four organic acids (lactic acid acetic acidpropionic acid and butyric acid) in saliva and tongue filmare shown in Table 3 Glucose (169 plusmn 105 120583gg in saliva 62plusmn 70 120583gg in film) and acetic acid (1458 plusmn 1215 120583gg in saliva3899 plusmn 2423 120583gg in film) were the predominant sugar andacid respectively

As shown in Table 3 the proportion of glucose wassignificantly lower (p lt 005) in film than in saliva while theproportions of fructose and sucrose were significantly higher(p lt 005) in film

The proportions of lactic and butyric acid were signifi-cantly lower (p lt 00001 for lactic acid p lt 001 for butyricacid) in film than in saliva while the proportion of acetic acidwas significantly higher (p lt 0001) in film (Table 3)

34 Concentrations of Cultivable Microorganisms The con-centrations in log

10(CFUg) of cultivable aerobic and anaero-

bic microorganisms in saliva and film are shown in Figure 2In saliva the mean concentration of aerobic microorgan-isms (210 times 107 CFUg) was slightly higher than that ofanaerobic microorganisms (156 times 107 CFUg) whereas infilm the mean concentrations of aerobic and anaerobicmicroorganisms were almost similar (194 times 108 CFUg and195 times 108 CFUg respectively) In other words the ratio ofanaerobicaerobic microorganisms was higher in film thanthat in saliva


Table 3 Concentrations and relative proportions (percentages) of sugars and organic acids in saliva and tongue filmThe p value representsthe level of significance when comparing the relative proportions of each metabolite between saliva and film (paired t-test)

Compound Concentration in Concentration in Proportion in Proportion in psaliva (120583ggplusmnSD) film (120583ggplusmnSD) saliva (plusmn SD) film (plusmn SD)

glucose 169 plusmn 105 62 plusmn 70 954 plusmn 105 897 plusmn 70 lt005fructose 03 plusmn 03 03 plusmn 02 16 plusmn 03 36 plusmn 02 lt005sucrose 01 plusmn 01 02 plusmn 03 04 plusmn 01 24 plusmn 03 lt005lactose 05 plusmn 03 03 plusmn 05 27 plusmn 03 43 plusmn 05 nslactate 150 plusmn 191 51 plusmn 31 109 plusmn 115 13 plusmn 09 lt0001acetate 1458 plusmn 1215 3899 plusmn 2423 676 plusmn 94 801 plusmn 71 lt00001propionate 449 plusmn 558 965 plusmn 969 162 plusmn 63 162 plusmn 63 nsbutyrate 126 plusmn 141 120 plusmn 151 54 plusmn 39 23 plusmn 29 lt001

Saliva-aerobic Saliva-anaerobic Tongue film-aerobic Tongue film-anaerobic

5

6

7

8

9

10

log(

CFU

g)

Figure 2 Box plot representation of cultivable aerobic and anaero-bic microorganisms counts in saliva and tongue film

35 Bacterial Communities in Saliva and Tongue Film Re-sults for the 5 phyla and 3 genera (Veillonella and Strepto-coccus belonging to Firmicutes and Prevotella belonging toBacteroidetes) quantified are shown in Figure 3 Overall themost abundant phyla were Bacteroidetes (377 plusmn 157 insaliva 196 plusmn 99 in film) and Firmicutes (92 plusmn 28 insaliva 110 plusmn 31 in film) Saliva samples were characterizedby significantly higher proportions of Bacteroidetes (plt0001)and its main genus Prevotella (plt001) whereas tongue filmssamples exhibited significantly higher proportions of Fir-micutes (plt005) Actinobacteria (p lt0001) and the genusStreptococcus (p lt 005)

36 Sensory Evaluation The mean sensitivity scores were41 (sweet) 35 (salty) 34 (sour) 31 (bitter) and 24(umami) respectively with a wide distribution across sub-jects (Figure 4(a)) In addition Figure 4(b) shows the biplotrepresentation of the principal component analysis (PCA)performed on the correlation matrix The first two factorsrepresented 89 of the initial variability of the data The F1axis explaining 76 of the variability clearly differentiatedsubjects according to their overall sensitivity indifferentlyfrom the taste considered On axis 2 the main oppositecontributors were scores of sensitivity to bitterness and

umami Accordingly significant correlations (p lt 001) wereobserved between sensitivity scores for all 5 tastes at theexception of scores for bitterness and umami (r = 0419)

37 Relationships between Taste Sensitivity Scores and Biolog-ical Variables Table 4 shows the correlations between sensi-tivity scores on the one hand and biological data organizedin 6 blocks (pH saliva flow or film weight concentrationsof acids concentrations of sugars total microbial count andproportions of the different bacterial phyla) on the otherhand To visualize the results correlations are classified asclose to null (0) weak (- or + for negative and positivecorrelations respectively) or moderate (-- or ++)

Given the size of the population studied it is not rea-sonable to focus on some specific correlations However thistable provides an interesting view of some trends linkingquite robustly some blocks and taste in general In particularsaliva flow was negatively correlated to sensitivity for mosttastes and although not standing for all tastes the negativetrend was also observed for weight of the tongue film overallsubjects with lower salivary flow and lower weights of filmperceived the tastes better There was a sharp contrast in theresults linking organic acids and sensitivity scores While thecorrelations were almost always positive (weak or moderate)in saliva they were close to null or weakly negative in tonguefilm Therefore concentrations of organic acids appearedmore influent in saliva than at proximity of the taste buds toexplain taste sensitivity

Looking at the sugars there was also a clear contrastbetween the results in film and in saliva This time a lotof correlations were close to null in saliva (15 ldquo0rdquo out of 20correlations) while only two ldquo0rdquo correlations were observedin tongue film In tongue film higher concentrations ofglucose and fructose were systematically associated withlower sensitivity while higher concentrations of sucrose andlactose were rather associated with higher sensitivity

Finally the last block where interesting and consistentcorrelations were found is the block of phyla proportionsIn saliva the proportions of Actinobacteria especially andFirmicutes to a slightly lesser extent were negatively associ-ated with sensitivity scores In tongue film the proportion ofBacteroidetes was positively associated with three tastes

Looking at the results from a different angle some tastesappeared associatedwithmore of the biological variables than


0

10

20

30

40

50

60

Firmicutes Bacteroidetes Proteobacteria Fusobacteria Actinobacteria

perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25

Veillonella Streptococcus0

10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 3 Composition of bacterial communities in saliva and tongue film (n=21) proportions of 5 phyla and 3 genera Significant differencebetween saliva and tongue film is indicated as follows lowast plt005 lowastlowast plt001 lowastlowastlowast plt 0001

others In particular it is interesting to note that the salty tasteshowed weak or moderate correlations for 14 out of the 16variables studied in saliva as opposed to only 716 for bitterand umami tastes for example In tongue film the taste thatshowed the most and the highest correlations was bitterness

4 Discussion

The objective of this work was to investigate whether the bio-logical film lining the tongue or tongue film may play a rolein taste perception For that purpose we first characterizedjointly tongue film and saliva of healthy subjects and foundthat these two biological fluids differed in composition andmicrobiological profiles Second it was evidenced that tastesensitivity in general was linked to some biological variablesin the two compartments but variables that appeared influentin saliva (flow organic acids proportion of Actinobacteriaand Firmicutes) and in tongue film (sugars and proportionsof Bacteroidetes) were not the same

The natural material found at the surface of the tongue ispoorly described except when it is present in excess (it is then

termed tongue coating) for example in patients sufferingfrom halitosis The tongue film characteristics found in thisstudy are slightly different from those of tongue coating Forexample pH of tongue coating was found to be more alkaline[15] than in the present study The weight of film sampledhere was lower than in another study [16] in which the meanweight was 173 mg (N=96) This first may be due to differentsampling procedures the tongue was scraped until no morecoating could be dislodged in vanTornout et al [16] while thetongue was scraped only once in this study Second subjectshad different oral health status in the two studies namelyhalitosis patients versus healthy subjects Other authors havereported an impact of oral diseases on tongue film quantitywith higher wet weight of tongue film in a periodontal diseasegroup (901 mg n=17) compared to the control group (146mg n=6) [17] The ratio of anaerobicaerobic bacteria intongue film here (close to 1) was also well below the value(229) found in tongue coating [18] However the authors alsoreported that the ratio decreased to 146 after treatment ofhalitosis by local antibiotics Again this highlights that the


Sweet Salty Sour Bitter Umami0

1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami

20 4 6minus4 minus2minus6F1 (759 )

minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)

Figure 4 Taste sensitivity scores Scattergram illustrating the distribution of scores across the panel (a) and biplot representation of theprincipal component analysis performed on the correlation matrix (b)

Table 4 Correlations between taste sensitivity scores and biological variables in saliva and in tongue film The Spearman correlationscoefficients are coded as follows -- for -05lt r lt -03 (moderate negative correlation) - for -03lt r lt -01 (weak negative correlation) 0for -01ltrlt01 + for 01lt rlt03 (weak positive correlation) ++ for 03lt r lt 05 (moderate positive correlation) A star indicates a significantcorrelation (plt005)

Salivary variables Sweet Salty Sour Bitter Umami Film variables Sweet Salty Sour Bitter UmamipH 0 + - + 0 pH 0 0 0 0 0flow - -- -- 0 --lowast weight - 0 - 0 --lactate ++ + + + + lactate - - 0 - 0acetate + ++ + + 0 acetate - 0 0 - 0propionate + ++lowast + + + propionate - 0 - 0 0butyrate ++ ++ + + 0 butyrate 0 0 0 0 -glucose 0 0 0 0 0 glucose - - - -- -fructose 0 ++ + 0 + fructose - - - -- --sucrose - - - 0 - sucrose ++ 0 + + +lactose 0 0 0 0 0 lactose + 0 + - +total bacterial count 0 + 0 0 0 total bacterial count - + - 0 0Actinobacteria -- --lowast - - -- Actinobacteria 0 0 - + -Bacteroidetes 0 + - 0 - Bacteroidetes + + 0 ++lowast 0Firmicutes -- - - - 0 Firmicutes 0 0 0 - -Proteobacteria 0 - 0 0 + Proteobacteria 0 - 0 0 +Fusobacteria 0 - 0 0 0 Fusobacteria 0 - - 0 0

available data for tongue coating do not correspond to thetongue film in a healthy situation

In terms of biochemical composition the concentra-tions found in saliva are consistent with previous reportsparticularly for glucose [19] and acetate [20] while toour knowledge no data is available for the organic acidsrsquocomposition of tongue film or tongue coating The origin ofthe organic acids in the oral cavity is repeatedly attributed tomicrobial metabolism This has been described particularlyfor the production of lactic acid from glucose or sucrosein saliva and in dental plaque [21 22] where the large

increase (5 to 8-fold change) in lactic acid concentration isboth rapid (within 5 minutes) and transient (returning tobasal level in approximately 30 minutes) However whenthe extracellular sugar supply is limited for example sometime away after a meal like in our conditions bacterialmetabolism is not dominated by lactate production but shiftsto production of mixed acids [23] Thus both in saliva [22]and in dental plaque [21] the predominant organic acidsin resting conditions are ordered as in the present studyie acetic acid gt propionic acid gt lactic acid gt butyricacid Our results indicate that the tongue film of healthy


individuals is comparatively richer in acetic acid than salivawhich may be linked to different bacterial communities Forexample Veillonella utilizes lactate specifically and producesacetate and propionate as end-products Other Firmicutessuch as Lactobacilli and Streptococci can also convert lactate toacetate as also Actinomyces belonging to the Actinobacteriaphylum [23] In our case we indeed found that the twophyla Firmicutes and Actinobacteria were more representedin tongue film

The 5 phyla quantified account for 80-95 of the entiresaliva microbiome [24] and are also abundant in other oralsamples [25 26] Our results differ from other reports inwhich Firmicutes was often the most abundant phylumin saliva [27] in tongue coating [28] or on the tonguedorsum [26] This divergence is likely linked to differentDNA extractionmethods but it does not prevent comparisonbetween subjects or correlations with taste sensitivity scores

Focusing now on the sensory results the correlativeapproach we chose in this study has been seldom appliedMost articles linking saliva properties and composition andtaste sensitivity have opted for an approach where groupswere constituted based on sensory results and differencesbetween groupswere tested [7ndash10]However one recent studycorrelated salivary biochemical data and taste sensitivity [29]Their main finding was that sweetness sensitivity correlatedwith salivary pH but only for 38 of subjects with the highestand lowest pH values Keeping all subjects in our study thelink between pH and sensitivity is not clearly establishedneither in saliva nor in tongue film

We found consistent negative correlations between tastesensitivity and saliva flowtongue film weight Concerningsaliva comparable correlations between flow and taste sensi-tivity have been previously described For example when anacid enters the oral cavity a high salivary flow (usually asso-ciated with higher buffering capacity of saliva) diminishesthe protons concentration at the receptors vicinity [6] andtherefore sensitivity would be lowered Similar associationswere reported between salivary flow and sensitivity to oleicacid [30] or NaCl perception [6] It is worth mentioning thatmethods used to determine taste sensitivity are most oftenperformed with tastants in solution which implies that thetaste solution is diluted in saliva present in the oral cavity Inour case the tastant is included in a solidmatrix placed on thetongue but the correlation also indicates that a low salivaryflow improves sensitivity It is therefore highly plausible thatthe amount of saliva on the tongue is related to saliva flow andthat at lowflow (yetwithin the healthy range) the local tastantconcentration is higher In addition the tongue film weightwas also negatively correlated to sensitivity translating thatthe film also acts as a barrier limiting the diffusion of the tastemolecules to the receptors on the tongueThis is for exampleconsistent with Quirynen et al [31] who found a loweringof taste identification threshold after mechanical cleansingof tongue treatment resulting in a lower index of tonguecoating The removal of tongue coating by mild brushing hasalso been previously shown to improve sour taste recognitionin older adults [12]

The correlation between taste sensitivity and organicacids concentrations were more numerous and stronger in

saliva than in tongue film and clearly indicate that highersalivary levels of organic acids are associated with a highersensitivity This relationship is intriguing and opposite to aprevious finding where higher organic acids concentrationsin saliva were associated with lower sensitivity to the tasteof fat [10] To date the mechanism linking organic acidsand taste sensitivity remains unknown In contrast to organicacids the correlation between taste sensitivity and sugarswere more numerous and stronger in tongue film than insaliva First it should be noted that contrarily to all theorganic acids which may be produced within the oral cavitythree of the four sugars measured are brought to the oralcavity through food intake (fructose found in fruit lactosefound in milk and sucrose produced from plant sources)It therefore seemed initially plausible that the variations inlevels of these sugars on the tongue film corresponded to thecapacity of naturally cleansing the tongue after food intakeHowever in the present study the signs of correlations weredifferent between taste and fructose on the one hand andtaste and lactose and sucrose on the other handThis suggeststhat the correlations donotmerely translate a generic capacityof tongue cleansing Another explanation may thereforereside in sugar metabolism found on the tongue dorsumThetwo sugars which tended to be positively associated with tastesensitivity were the disaccharides sucrose and lactose whilethe monosaccharides fructose and glucose were negativelyassociated with taste sensitivity In mammalians the disac-charides can be hydrolyzed by intestinal enzymes and mostoral bacteria can also use disaccharides for glycolysis Perhapsmore interestingly sucrose specifically can be converted byoral bacteria into glucan and fructan which serve as ldquobuild-ing materialrdquo for biofilms [23] This phenomenon has beenmainly described for Streptococci in the context of bacterialadhesion to the dental surfaces [32] but glycoproteins havealso been observed surrounding bacteria adherent tomucosalcells of tongue rats [33] If such glycoproteins are at leastpartly of bacterial origin this would mean that the sucroseconversion to glucanfructan participates in the strength ofbiofilms on the tongue surface Therefore a higher level ofsucrose in the tongue filmmay indicate a lower proportion ofbacteria capable of converting them into glucan and fructan(or a lower conversion rate) and therefore a less firmlystructured biofilmThe physical barrier between tastants andtaste receptors would as a consequence be less efficient andsensitivity increased A finer characterization of the bacterialgenera and species in the tongue film and targeted studyof their sugar metabolisms would be necessary to test thishypothesis This conclusion also stands when looking at thebacteriological results at present it is suggested that higherproportions of Actinobacteria and Firmicutes in saliva arelinked to lower taste sensitivity while a higher proportion ofBacteroidetes in the tongue film increases sensitivity (partic-ularly to bitterness) Given the diversity of genera and specieswithin a phylum described elsewhere in detail for the oralmicrobiome [34] it is overall difficult to propose mechanisticexplanations on those links This is especially true for theoral cavity where particularly high within-subject diversitywas reported [26] Again a more detailed characterization ofmicrobial communities and of their metabolism would be of


interest but this study demonstrates that the oralmicrobiomedeserves to be considered as an explanatory variable wheninvestigating perireceptor events involved in taste perceptionTo conclude one should keep in mind that only a fewcorrelations between taste sensitivity scores and biologicalvariables were significant and such correlationsweremodestIn addition this preliminary study was conducted on alimited number of subjects and should be extended to a largerpanel Nevertheless in spite of such limitations this workalready points to two interesting areas in taste research theoral microbiome in general and the specific characterizationof the film lining the tongue

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author on reason-able request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors gratefully acknowledge the China ScholarshipCouncil (CSC) for its financial support

References

[1] L Brondel A Jacquin S Meillon and L Penicaud ldquoTastePhysiology roles and dysfunctionrdquo Nutrition Clinique etMetabolisme vol 27 no 3 pp 123ndash133 2013

[2] O Hershkovich and R M Nagler ldquoBiochemical analysis ofsaliva and taste acuity evaluation in patients with burningmouth syndrome xerostomia andor gustatory disturbancesrdquoArchives of Oral Biolog vol 49 no 7 pp 515ndash522 2004

[3] K Solemdal L Sandvik T Willumsen M Mowe and THummel ldquoThe impact of oral health on taste ability in acutelyhospitalized elderlyrdquo PLoS ONE vol 7 no 5 Article ID e365572012

[4] W Skrandies and R Zschieschang ldquoOlfactory and gustatoryfunctions and its relation to body weightrdquo Physiology amp Behav-ior vol 142 pp 1ndash4 2015

[5] M Y Pepino S Finkbeiner G K Beauchamp and J A Men-nella ldquoObese women have lower monosodium glutamate tastesensitivity and prefer higher concentrations than do normal-weight womenrdquo Obesity vol 18 no 5 pp 959ndash965 2010

[6] R Matsuo ldquoRole of saliva in the maintenance of taste sensitiv-ityrdquo Critical Reviews in Oral Biology and Medicine vol 11 no 2pp 216ndash229 2000

[7] M Dsamou O Palicki C Septier et al ldquoSalivary proteinprofiles and sensitivity to the bitter taste of caffeinerdquo ChemicalSenses vol 37 no 1 pp 87ndash95 2012

[8] T Cabras M Melis M Castagnola et al ldquoResponsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels oftwo specific basic proline-rich proteins in humansrdquo PLoS ONEvol 7 no 2 Article ID e30962 2012

[9] L Rodrigues G da Costa C Cordeiro C C Pinheiro FAmado and E Lamy ldquoRelationship between saliva protein

composition and 6-n-Propylthiouracil bitter taste responsive-ness in young adultsrdquo Journal of Sensory Studies vol 32 no 4Article ID e12275 2017

[10] R Mounayar M Morzel H Brignot et al ldquoSalivary markersof taste sensitivity to oleic acid A combined proteomics andmetabolomics approachrdquoMetabolomics vol 10 no 4 pp 688ndash696 2014

[11] M M Danser S M Gomez and G A Van der WeijdenldquoTongue coating and tongue brushing a literature reviewrdquoInternational Journal of Dental Hygiene vol 1 no 3 pp 151ndash1582003

[12] T Ohno H Uematsu S Nozaki and K Sugimoto ldquoImprove-ment of taste sensitivity of the nursed elderly by oral carerdquoJournal of Medical and Dental Sciences vol 50 no 1 pp 101ndash107 2003

[13] K Solemdal L Sandvik T Willumsen M Mowe and THummel ldquoThe impact of oral health on taste ability in acutelyhospitalized elderlyrdquo PLoS ONE vol 7 p 7 no 5 Article IDe36557 2012

[14] E Zaura B W Brandt A Prodan et al ldquoOn the ecosystemicnetwork of saliva in healthy young adultsrdquo The ISME Journalvol 11 no 5 pp 1218ndash1231 2017

[15] E D S Tolentino L E M Chinellato and O Tarzia ldquoSalivaand tongue coating pH before and after use of mouthwashesand relationshipwith parameters of halitosisrdquo Journal of AppliedOral Science vol 19 no 2 pp 90ndash94 2011

[16] M Van Tornout J Dadamio W Coucke and M QuirynenldquoTongue coating Related factorsrdquo Journal of Clinical Periodon-tology vol 40 no 2 pp 180ndash185 2013

[17] K Yaegaki and K Sanada ldquoVolatile sulfur compounds inmouth air from clinically healthy subjects and patients withperiodontal diseaserdquo Journal of Periodontal Research vol 27 no4 pp 233ndash238 1992

[18] E H De Boever and W J Loesche ldquoAssessing the contributionof anaerobic microflora of the tongue to oral malodorrdquo TheJournal of the American Dental Association vol 126 no 10 pp1384ndash1393 1995

[19] M J A Campbell ldquoGlucose in the saliva of the non-diabeticand the diabetic patientrdquo Archives of Oral Biolog vol 10 no 2pp 197ndash205 1965

[20] Z-F Chen BWDarvell andVW-H Leung ldquoHuman salivaryanionic analysis using ion chromatographyrdquo Archives of OralBiolog vol 49 no 11 pp 863ndash869 2004

[21] D A M Geddes ldquoAcids produced by human dental plaquemetabolism in siturdquo Caries Research vol 9 no 2 pp 98ndash1091975

[22] Y-D Park J-H Jang Y-J Oh and H-J Kwon ldquoAnalyses oforganic acids and inorganic anions and their relationship inhuman saliva before and after glucose intakerdquo Archives of OralBiolog vol 59 no 1 pp 1ndash11 2014

[23] N Takahashi ldquoOral microbiome metabolism From ldquowho aretheyrdquo to ldquowhat are they doingrdquordquo Journal of Dental Researchvol 94 no 12 pp 1628ndash1637 2015

[24] H Chen and W Jiang ldquoApplication of high-throughputsequencing in understanding human oral microbiome relatedwith health and diseaserdquo Frontiers in Microbiology vol 5 2014

[25] E M Bik C D Long G C Armitage et al ldquoBacterial diversityin the oral cavity of 10 healthy individualsrdquo The ISME Journalvol 4 no 8 pp 962ndash974 2010

[26] CHuttenhower DGevers R Knight et al ldquoStructure functionand diversity of the healthy human microbiomerdquo Nature vol486 no 7402 pp 207ndash214 2012


[27] S J S Cameron S AHuwsM JHegarty D PM Smith and LA J Mur ldquoThe human salivary microbiome exhibits temporalstability in bacterial diversityrdquo FEMS Microbiology Ecology vol91 no 9 Article ID fiv091 2015

[28] B Jiang X Liang Y Chen et al ldquoErratum Integrating next-generation sequencing and traditional tongue diagnosis todetermine tongue coating microbiomerdquo Scientific Reports vol2 2012

[29] K-I Aoyama Y Okino H Yamazaki et al ldquoSaliva pH affectsthe sweetness senserdquo Nutrition Journal vol 35 pp 51ndash55 2017

[30] E Neyraud O Palicki C Schwartz S Nicklaus and G FeronldquoVariability of human saliva composition possible relationshipswith fat perception and likingrdquo Archives of Oral Biolog vol 57no 5 pp 556ndash566 2012

[31] MQuirynen P Avontroodt C SoersH ZhaoM Pauwels andD Van Steenberghe ldquoImpact of tongue cleansers on microbialload and tasterdquo Journal of Clinical Periodontology vol 31 no 7pp 506ndash510 2004

[32] F Zhu H Zhang and H Wu ldquoGlycosyltransferase-mediatedsweet modification in oral streptococcirdquo Journal of DentalResearch vol 94 no 5 pp 659ndash665 2015

[33] I-S Watanabe K Ogawa D P Cury et al ldquoFine structureof bacterial adhesion to the epithelial cell membranes of thefiliform papillae of tongue and palatine mucosa of rodents Amorphometric TEM and HRSEM studyrdquoMicroscopy Researchand Technique vol 76 no 12 pp 1226ndash1233 2013

[34] F E Dewhirst T Chen J Izard et al ldquoThe human oralmicrobiomerdquo Journal of Bacteriology vol 192 no 19 pp 5002ndash5017 2010

Hindawiwwwhindawicom

International Journal of

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Anatomy Research International

PeptidesInternational Journal of



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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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BioinformaticsAdvances in

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Cell BiologyInternational Journal of



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ArchaeaHindawiwwwhindawicom Volume 2018


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MicrobiologyHindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom

Research ArticleThe Associations between Biochemical and MicrobiologicalVariables and Taste Differ in Whole Saliva and in the FilmLining the Tongue

Yunzi Feng12 Heacutelegravene Licandro3 Christophe Martin2 Chantal Septier2 Mouming Zhao 1

Eric Neyraud 2 andMartine Morzel2

1School of Food Science and Engineering South China University of Technology Guangzhou 510640 China2Centre des Sciences du Gout et de lAlimentation AgroSup Dijon CNRS INRA Universite de Bourgogne Franche-Comte21000 Dijon France3UMR A 02102 Procedes Alimentaires et Microbiologiques (PAM) AgroSup Dijon Universite de Bourgogne Franche-Comte21000 Dijon France

Correspondence should be addressed to Eric Neyraud ericneyraudinrafr

Received 10 January 2018 Accepted 21 May 2018 Published 14 June 2018

Academic Editor Asta Tvarijonaviciute

Copyright copy 2018 Yunzi Feng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this work was to investigate whether the biological film lining the tongue may play a role in taste perception Forthat purpose the tongue film and saliva of 21 healthy subjects were characterized focusing on microorganisms and their mainmetabolic substrates and products In parallel taste sensitivity was evaluated using a test recently developed by our group and thelinks between biological and sensory data were explored by a correlative approach Saliva and tongue film differed significantly inbiochemical composition (proportions of glucose fructose sucrose and lactic butyric and acetic acids) and in microbiologicalprofiles compared to saliva tongue film was characterized by significantly lower proportions of Bacteroidetes (plt0001) and itsmain genus Prevotella (plt001) and significantly higher proportions of Firmicutes (plt001) Actinobacteria (plt0001) and thegenus Streptococcus (plt005) Generic taste sensitivity was linked to biological variables in the two compartments but variablesthat appeared influent in saliva (flow organic acids proportion of Actinobacteria and Firmicutes) and in tongue film (sugars andproportions of Bacteroidetes) were not the same This study points to two interesting areas in taste research the oral microbiomeand the specific characterization of the film lining the tongue

1 Introduction

Eating behavior is a key factor of health in humans someof the major pathologies affecting the modern societies suchas obesity cardiovascular diseases or type 2 diabetes havebeen reported to be strongly linked to it Determinantsof eating behavior are various and comprise for examplebiological psychological and socioeconomic factors Amongbiological factors the sense of taste participates in thesensory perception of the food and thus influences foodchoices Impairments in taste perception can for examplelead to eating disorders and malnutrition [1] Probably themost objective way of characterizing and classifying subjectsaccording to their taste function is to measure their taste

sensitivity and more precisely their individual detectionthreshold This approach is thus widely used in patients withoral complaints [2 3] or with diet-related conditions such asobesity [4 5]

Taste perception occurs after activation of specializedreceptors in the taste buds on the tongue Saliva is theprincipal fluid that interacts with food and it is the mediumthat bathes the taste buds thus it plays an important role intaste perception through several mechanisms such as protec-tion of the taste receptors or transport of taste compounds[6] Several studies have indeed described the relationshipbetween salivary composition and taste sensitivity for exam-ple to bitterness [7ndash9] or to the taste of oleic acid [10] Besidesfree-flowing saliva the biological film lining the tongue

HindawiBioMed Research InternationalVolume 2018 Article ID 2838052 10 pageshttpsdoiorg10115520182838052

















2) at 37∘C for 48









SD deviation(SD)






3 Results














5

6

7

8

9

10

log(

CFU

g)











0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















2) at 37∘C for 48









SD deviation(SD)






3 Results














5

6

7

8

9

10

log(

CFU

g)











0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









2) at 37∘C for 48









SD deviation(SD)






3 Results














5

6

7

8

9

10

log(

CFU

g)











0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





SD deviation(SD)






3 Results














5

6

7

8

9

10

log(

CFU

g)











0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







5

6

7

8

9

10

log(

CFU

g)











0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

10

20

30

40

50

60


perc

entage

()

perc

entage

()

perc

entage

()

SalivaFilm

0

5

10

15

20

25


10

20

30

40

Prevotella

lowast

lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion






1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




1

2

3

4

5

6

(a)

Sweet

SaltySour

Bitter

Umami


minus6

minus4

minus2

0

2

4

6

F2 (1

28

)(b)
















Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Data Availability




Acknowledgments


References







































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

The Associations between Biochemical and Microbiological … · 2021. 1. 14. · ResearchArticle The Associations between Biochemical and Microbiological Variables and Taste Differ