Chemosensate-Induced Modulation of the Salivary Proteome and

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Chemistry and Biology of Aroma and Taste

Chemosensate-Induced Modulation of the Salivary Proteome and Metabolome Alters the Sensory Perception of Salt Taste and Odor-Active Thiols Matthias Bader, Theresa Stolle, Maximilian Jennerwein, Jürgen Hauck, Buket Sahin, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02772 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Journal of Agricultural and Food Chemistry

1

Chemosensate-Induced Modulation of the Salivary

2

Proteome and Metabolome Alters the Sensory

3

Perception of Salt Taste and Odor-Active Thiols

4 5

Matthias Bader†, Theresa Stolle†, Maximilian Jennerwein†, Jürgen Hauck†,

6

Buket Sahin‡ and Thomas Hofmann†‡§*

7 †

8

University of Munich, Lise-Meitner-Straße 34, D-85354 Freising, Germany,

9

‡

Leibniz-Institute for Food Systems Biology at the Technical University of

10

Munich, Lise-Meitner Str. 34, D-85354 Freising, Germany,

11 12

Chair for Food Chemistry and Molecular Sensory Science, Technical

§

Bavarian Center for Biomolecular Mass Spectrometry, Technical University of Munich, Gregor-Mendel-Straße 4, 85354 Freising, Germany.

13 14 15 16 17

*

To whom correspondence should be addressed

18

PHONE

+49-8161/71-2902

19

FAX

+49-8161/71-2949

20

E-MAIL

[email protected]

21 22 23

1 Environment ACS Paragon Plus


24

ABSTRACT

25 26

Oral stimulation with chemosensates were found to trigger changes in the

27

composition of the salivary proteome and metabolome which translate into a

28

functional modulation of odor and taste perception. Orosensory intervention

29

with 6-gingerol induced a significant increase of the abundance of the salivary

30

sulfhydryl oxidase 1 which was found to catalyze the oxidative decline of the

31

odor-active 2-furfurylthiol, thus resulting in a decrease of the odorant levels in

32

the exhaled breath as shown by PTR-MS and a reduction of the perceived

33

sulfury after-smell. Therefore, the sulfhydryl oxidase 1 may be considered as

34

a component of a molecular network triggering oral cleansing mechanisms

35

after food ingestion. Moreover, oral stimulation with citric acid, followed by

36

targeted metabolomics was found to induce a strong increase of salivary

37

concentrations of minerals and, in particular, sodium ions, while the other

38

metabolites were rather unaffected.

39

salivary sodium after citric acid stimulation, NaCl test stimuli were perceived

40

as significantly less salty, most likely due to the decreased sensory contrast.

41

This indicates the modulation of the salivary proteome and metabolome to be

42

a major peri-receptor event in fine-tuning odor and taste sensitivity.

43

KEYWORDS: saliva, metabolome, 2-furfurylthiol, PTR-MS, taste, peri-

44

receptor, salt taste

Due to the elevated basal levels of

45


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46

INTRODUCTION

47 48

Healthy humans show an average daily flow of 1.0 to 1.5 L saliva secreted as

49

a clear mucoserous body fluid from the paired major submandibular,

50

sublingual, and parotid glands, as well as a series of minor salivary glands.1,2

51

Affected by several factors like diet, age, day time, gender, as well as health

52

state,2 saliva consists of a truly complex mixture of electrolytes, small organic

53

molecules, peptides, and proteins contributing to the saliva’s buffer capacity,2-

54

4

55

of the oral mucosal structure via both direct antimicrobial action and

56

agglutination or surface exclusion of microbes.6,7

the acquired pellicle formation on the teeth surface,5-7 and immune defense

57

Although a total number of 1166 proteins could be identified in human

58

saliva within the last decade,8-18 the recent assignment of the entire human

59

proteome indicates the presence of more than 7400 proteins in saliva.19

60

Various functions could be assigned to distinct salivary proteins, e.g. mucins

61

contribute to bolus formation and lubrication during mastication,1 cooperate

62

with calcium and phosphate on teeth mineralization and protection from acid-

63

derived demineralization,2 and are key to generate the pellicle on the tooth

64

surface.5,20 Moreover, salivary proteins play a key role in food digestive

65

processes, e.g. salivary α-amylase initiates

66

breakdown which has been correlated to an increased salt perception of

67

starch-based food products.21 While salivary histatins and proline-rich proteins

68

have been demonstrated to complex astringent polyphenols and, by doing so,

69

to inhibit irritations of the gastric system,22-24 lipases such as, e.g. lipase K, M,

70

and N, excreted from the von Ebner glands were reported to hydrolyze dietary

an early-stage amylose



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71

triglycerides to release free fatty acids which in turn activate fatty acid

72

responsive receptors like GPR 120 in taste buds.25 Intriguingly, lipolytic

73

activities detected in minor salivary gland secretions directly supplying

74

gustatory papillae were correlated to individual sensitivities for triglycerides,

75

suggesting that differential lipase levels may contribute to variant fat

76

perception.25

77

Oral stimulation with basic taste compounds as well as trigeminal

78

chemosensates were not only shown to increase saliva flow, but also to

79

induce a massive change in saliva proteome composition.26-30 Very recently,

80

tryptic digestion of saliva samples collected after stimulation with citric acid

81

(sour) and 6-gingerol (pungent), followed by nano-HPLC-MS/MS, label-free

82

protein quantitation, and gene ontology enrichment analysis showed evidence

83

for stimulus-induced alterations of the saliva proteome to trigger an efficient

84

molecular

85

lactoperoxidase, myeloperoxidase, and lysozyme.30 Moreover, an increased

86

abundance of salivary sulfhydryl oxidase 1 was observed upon oral

87

stimulation with 6-gingerol.30 Although sulfhydryl oxidases are known to

88

catalyze the oxidation of mercaptans giving rise to the corresponding

89

disulfides31-33 and mercaptans, such as, e.g. the coffee key odorant 2-

90

furfurylthiol,34 has been reported to be partially degraded in the presence of

91

saliva,35,36 it is unclear whether or not an increase in salivary sulfhydryl

92

oxidase 1 activity translates into an altered sensory perception of odor-active

93

mercaptans.

defense

network

of

the

oral

cavity

involving

salivary

94

Next to the saliva proteome, also the saliva metabolome has been

95

shown to be affected upon oral stimulation with taste compounds.37-39


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96

However, any systematic and comparative data on the impact of various taste

97

stimuli on the salivary metabolome are lacking. In particular, it is yet unclear

98

as to whether and, if so, which tastant-induced changes in saliva proteome

99

and metabolome may translate into an altered sensitivity for odor and taste

100

perception.

101

The objectives of the present investigation were, first, to answer the

102

question as to whether an increased abundance of salivary sulfhydryl oxidase

103

1, recently observed upon oral stimulation with 6-gingerol,30 affects the

104

sensory perception of odor-active mercaptans, using the key food odorant 2-

105

furfurylthiol as a representative example. Second, salivary metabolome

106

alterations, induced upon oral stimulation with taste and trigeminal stimuli,

107

respectively, should be quantitatively monitored and metabolite changes

108

observed should be studied for their relevance in modulating taste perception.

109 110 111 112

MATERIALS AND METHODS

113

Chemicals. The following compounds were obtained commercially: L-

114

amino acids, nucleosides, nucleotide phosphates, organic acids, inorganic

115

salts, α-amylase (from hog pancreas, 50 U/mg), mucin (from bovine

116

submaxillary glands), ammonium acetate solution (5 mol/L), formic acid,

117

aspartame, citric acid, 6-gingerol, sodium chloride, 2-furfurylthiol, furfuryl

118

sulfide,

119

ammonium hydroxide solution (25%; Fluka, Neu-Ulm, Germany). Stable

120

isotope labeled amino acids and organic acids were purchased from

and

furfuryl

disulfide

(Sigma-Aldrich,


Steinheim,

Germany),


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121

Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA), stable isotope

122

labeled nucleotides were from Silantes (München, Germany). Potassium

123

bicarbonate, potassium chloride, sodium carbonate, and mono sodium L-

124

glutamate were obtained from Merck (Darmstadt, Germany). Water for

125

chromatographic separations was purified with an integral 5 system (Millipore,

126

Schwalbach, Germany), and acetonitrile for LC-MS analysis was obtained

127

from J.T.Baker (Deventer, Netherlands). Bottled water (Evian) was used for

128

sensory analyses. A purified mixture of iso-α-acids (purity >98%) was isolated

129

from

130

Hopfenveredelungsgesellschaft mbH, Mainburg, Germany), hydroxy-α- and

131

hydroxy-β-sanshool (purity >98%, each) were isolated from Zanthoxylum

132

piperitum as reported earlier.40

a

commercial

iso-α-extract

(30%;

Hallertauer

133

Chemosensory Stimulation and Collection of Saliva Samples. Eight

134

healthy volunteers (4 female, 4 male, ages 24-31), recruited from the

135

Technische Universität München and giving informed written consent to the

136

work, were asked to brush their teeth and rinse their mouth with water (100

137

mL) after breakfast and, with the exception of water, not to consume any food

138

products and smoking articles, respectively, for 2 h prior to the experiment.

139

Following a protocol reported earlier,30 non-stimulated and stimulated

140

saliva samples were collected as follows: to collect non-stimulated saliva

141

(control), the volunteers were asked to rinse their oral cavity with water (8 mL)

142

for 60 s and, then, to spit out. After waiting for 60 s and swallowing, water (4

143

mL) was taken into the mouth and, after performing chewing motions for 30 s,

144

the subjects were asked to expectorate in a pre-weighed petri dishes (pre-

145

stimulus sample, t0). For the sampling of stimulated saliva, the subjects were


Page 7 of 43


146

asked to rinse their mouth with water (8 mL) and, then, to spit out. After

147

waiting for 60 s and swallowing, an aliquot (4 mL) of water (control) or an

148

aqueous stimulus solution of citric acid (stimulus S1; 156 mmol/L), aspartame

149

(stimulus S2; 3.4 mmol/L), iso-α-acids (stimulus S3; 0.3 mmol/L), mono

150

sodium L-glutamate (stimulus S4; 30 mmol/L), sodium chloride (stimulus S5;

151

513 mmol/L), 6-gingerol (stimulus S6; 1.7 mmol/L), hydroxy-α-sanshool

152

(stimulus S7; 4 mmol/L), and hydroxy-β-sanshool (stimulus S8; 4 mmol/L),

153

respectively, was taken up in the mouth and, after performing chewing

154

motions for 15 s, the subjects were asked to expectorate in a pre-weighed

155

petridishes (stimulus sample t15). Thereafter, the subjects were asked not to

156

swallow and to take a sample of bottled water (4 mL) into the mouth and to

157

chew for 30 s and then to expectorate again (post-stimulus sample t45).

158

Without swallowing in between, the later part of this experiment was repeated

159

once to afford a second post-stimulus sample (t75). Thereafter, an aliquot (100

160

µL) of a LiCl solution (237 mmol/L) was added as an internal standard to each

161

saliva sample collected in order to determine the saliva volume via analysis of

162

the Li+ concentration in each sample as detailed below. Triplicates were

163

collected from each sample and were stored at -80° C until further analysis.

164

Quantitation of Anions and Cations in Saliva. Aliquots (100 µL) of

165

the saliva samples (t0, t15, t45, t75) were mixed with acetonitrile (300 µL),

166

vortexed for 10 s, and centrifuged (13400 rpm, 4° C) for 10 min to afford the

167

supernatant

168

acetonitrile/water (70/30, v/v; 300 µL) and again centrifuged (13400 rpm, 4° C,

169

10 min) to separate the supernatant. The corresponding supernatants were

170

combined, dried under a stream of nitrogen, taken up in water (500 µL) and,

and

the

protein

pellet,

which


was

re-suspended

in


171

then, anions and cations were analyzed by means of high-performance ion

172

chromatography.

173

The analysis of chloride, phosphate, sulfate, formate, and acetate in

174

saliva was performed on a Dionex IC 5000 system (Dionex, Idstein, Germany)

175

consisting of a Dionex ICS-5000 DP Dual Pump, a AS-AP autosampler, a

176

ICS-5000 CD digital conductivity detector (Dionex ASR Ultra suppressor cell),

177

and a EGC-KOH Cartridge eluent generator. Anions were separated on a 250

178

x 2 mm, 4 µm, Dionex IonPacTM AS11-HC (Dionex, Idstein) with EGC KOH as

179

eluent and the following gradient (flow rate: 0.015 mL/min): 1 mmol/L isocratic

180

for 8 min, within 10 min to 15 mmol/L, within 10 min to 30 mmol/L, within 10

181

min to 60 mmol/L, then isocratic for 7 min and, finally back to 1 mmol/L in 2

182

min, followed by a column equilibration phase for 13 min.

183

For analysis of ammonium, sodium, potassium, magnesium, calcium,

184

and lithium ions, the later were added as internal standard to calculate the

185

saliva volume, the Dionex ICS-2000 apparatus was used with a digital

186

conductivity detector, a CSRS 300 suppressor cell, an AS autosampler, and

187

an eluent generator equipped with a RFIC EluGen cartridge EGC III MSA

188

(Dionex). Cations were separated with a 250 x 2 mm, 4 µm, Dionex IonPacTM

189

CS19 RFICTM (Dionex, Idstein) using 5 mmol/L MSA for isocratic elution with

190

a flow rate of 0.25 mL/min. Data analysis was performed using Chromeleon

191

software 6.80.

192

For method validation, aliquots (400 µL) of saliva were spiked with an

193

aliquot (100 µL) of diluted stock solutions containing the target cations or

194

anions in triplicate and further processed as described before. As control, an

195

aliquot of the artificial saliva (400 µL) was mixed with water (100 µL) instead


Page 8 of 43

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196

of the target analytes and further processed as mentioned above (see Table

197

S1).

198

Quantitation of Free Amino Acids in Saliva. After centrifugation

199

(13400 rpm, 4° C, 10 min), aliquots (500 µL) of saliva samples were mixed

200

with an aliquot (10 µL) of an internal standard solution containing the stable

201

isotope labeled amino acids

202

µmol/L),

203

13

204

L-glutamic

205

(729 µmol/L), 1,2-13C2-L-leucine (901 µmol/L),

206

2

207

serine (953 µmol/L),

208

µmol/L), 2H4-L-tyrosine (578 µmol/L), and

209

equilibration (15 min), an aliquot (490 µL) of ice-cold acetonitrile was added,

210

the sample mixtures were vortexed (10 s), centrifuged (13400 rpm, 4° C, 10

211

min) and, then, aliquots (2 µL) of the supernatants were analyzed by means of

212

UPLC-MS/MS. UPLC-MS/MS analysis was performed using an Acquity i-class

213

core system (Waters, Milford, MA, USA) consisting of a binary solvent

214

manager, sample manager and column oven connected to a Waters Xevo

215

TQ-S mass spectrometer (Waters, Manchester, UK). Chromatographic

216

separation was performed on a 150 × 2.1 mm i.d., 1.7 µm, BEH Amide

217

column (Waters, Eschborn, Germany) using a binary gradient of solvent A

218

consisting of an aqueous ammonium acetate solution (5 mmol/L) adjusted to

219

pH 3.0 with formic acid and solvent B consisting of mixture (95/5, v/v)

220

acetonitrile and ammonium acetate (5 mmol/L; pH 3.0). Chromatography was

13

13

C2-glycine (1319 µmol/L),

C6-L-arginine (587 µmol/L),

C415N-L-aspartic acid (710 µmol/L), acid (679 µmol/L),

13

C3-L-alanine (1151

N2-L-asparagine (1367 µmol/L),

C5-L-glutamine (959 µmol/L),

C6-L-histidine (837 µmol/L),

H5-L-phenylalanine (599 µmol/L), 13

13

15

13

13

13

13

C515N-

13

C6-L-isoleucine

C615N2-L-lysine (473 µmol/L),

C515N-L-proline (982 µmol/L),

13

C3-L-

C415N-L-threonine (838 µmol/L), 2H5-L-tryptophane (492 13

C5-L-valine (812 µmol/L). After



Page 10 of 43

221

performed at a flow rate of 0.4 mL/min with an initial mixture of 10% solvent A

222

and 90% solvent B for 1 min. Thereafter, solvent A was increased within 5.5

223

min to 34.4 %, thereafter in 2.5 min to 100 % and, then, kept for 2.5 min.

224

Finally, the starting conditions were adjusted in 0.5 min and kept for 2 min.

225

The concentrations were calculated using response curves for each

226

amino acid determined by analysis of defined amounts of labeled and

227

unlabeled amino acids in different concentration ratios (from 0.002 to 3.3

228

mg/L; eight-point calibration). Using the multiple reaction monitoring (MRM)

229

mode operating in the positive ionization mode (ESI+), amino acids were

230

analyzed in the effluent after tuning the MS parameters for each compound.

231

The following mass transitions were used for the analytes and the

232

corresponding internal standards: glycine (m/z 75.7→75.8)/

233

78.8→79.0), L-alanine (m/z 90.1→44.1)/

234

arginine (m/z 175.2→116.1)/

235

acid (m/z 134.1→74.1)/

236

glutamine (m/z 147.1→84.1)/

237

acid (m/z 148.1→84.1)/

238

(m/z 156.1→110.1)/

239

132.1→86.1)/

13

240

132.1→86.1)/

1,2-13C2-L-leucine

241

147.2→84.1)/

242

166.1→120.1)/

2

243

116.1→70.1)/

13

244

106.0→60.1)/

245

13

13

13

13

13

13

13

13

C2-glycine (m/z

C3-L-alanine (m/z 92.85→46.1), L-

C6-L-arginine (m/z 181.0→74.1), L-aspartic

C415N-L-aspartic acid (m/z 139.2→77.0), L13

C5-L-glutamine (m/z 152.2→88.1), L-glutamic

C515N-L-glutamic acid (m/z 154.2→89.1), L-histidine

C6-L-histidine (m/z 161.9→115.1), L-isoleucine (m/z

C6-L-isoleucine

(m/z (m/z

138.2→91.1), 134.1→88.1),

L-leucine

(m/z

L-lysine

(m/z

C615N2-L-lysine (m/z 155.3→90.1), L-phenylalanine (m/z H5-L-phenylalanine (m/z 170.9→125.1), L-proline (m/z C515N-L-proline

(m/z

122.2→75.1),

L-serine

(m/z

13

C3-L-serine (m/z 108.8→62.0), L-threonine (m/z 120.1→74.1)/

C415N-L-threonine (m/z 125.2→78.1), L-tryptophan (m/z 205.1→188.0)/ 2H5-


Page 11 of 43


(m/z 209.9→150.1), L-tyrosine (m/z 182.1→136.1)/

2

246

L-tryptophane

247

tyrosine (m/z 186.0/140.1), and L-valine (m/z 118.15→72.1)/

248

(m/z 124.2→77.1). Taurine (m/z 125.8→108.0) was quantitated via

249

threonine (m/z 125.2→78.1), L-citrulline (m/z 176.02→70.05) via

13

C6-L-

250

arginine (m/z 181.0→74.1), L-ornithine (m/z 132.9→69.9) via

15

N2-L-

251

asparagine

252

108.8→62.0) as the corresponding internal standard.

(m/z

135.2→89.1),

and

choline

via

13

H4-L-

C5-L-valine 13

C415N-L-

13

C3-L-serine

(m/z

253

The Xevo TQ-S mass spectrometer operated in positive electrospray

254

(ESI+). The instrument parameters were set as follows: capillary voltage (2.5

255

kV), sampling cone (22 V), source offset (50 V), source temperature (150° C),

256

desolvation temperature (400° C), cone gas (150 L/h), desolvation gas (800

257

L/h), collision gas flow (0.15 mL/min) and nebulizer gas (7 bar). Data was

258

processed and analyzed using MassLynx 4.1 and Targetlynx (Waters,

259

Manchester, UK).

260

Quantitation of Organic Acids, Nucleosides, and Nucleotide

261

Phosphates in Saliva. After centrifugation (13400 rpm, 4° C, 10 min),

262

aliquots (125 µL) of the saliva samples were mixed with an internal standard

263

solution (10 µL) of the isotope-labeled organic acids

264

µmol/L) and 2H4-succinic acid (794 µmol/L) and an internal standard solution

265

(25 µL) of isotope-labeled nucleotide phosphates

266

GTP (50 µmol/L),

267

equilibration (15 min), ice-cold acetonitrile (90 µL) was added, the sample

268

mixtures were vortexed (10 s) and centrifuged (13400 rpm, 4° C, 10 min) and,

269

then, aliquots (2 µL) of the supernatants were analyzed by UPLC-MS/MS.

270

UPLC-MS/MS analysis was performed using an Acquity i-class core system

15

N5-GMP (49 µmol/L) and

15

15

15

N2-uric acid (1000

N5-ATP (50 µmol/L),

15

N5-

N5-AMP (51 µmol/L). After



271

(Waters, Milford, MA, USA) consisting of a binary solvent manager, sample

272

manager and column oven connected to a Waters Xevo TQ-S mass

273

spectrometer (Waters, Manchester, UK). Chromatographic separation was

274

performed on a 100 × 2,1 mm i.d., 5 µm, SeQuant ZIC-pHILIC (Merck,

275

Darmstadt, Germany) using a binary gradient with an aqueous ammonium

276

acetate solution (5 mmol/L), adjusted to pH 9.5 with ammonium hydroxide

277

solution, as solvent A and a mixture (95/5, v/v) of acetonitrile and ammonium

278

acetate (5 mmol/L), adjusted to pH 9.5 with ammonium hydroxide solution, as

279

solvent B. Chromatography was performed at a flow rate of 0.3 mL/min with

280

an initial mixture of 20% solvent A and 80% solvent B for 3.5 min. Thereafter,

281

solvent A was increased within 3.5 min to 25 %, thereafter in 5.5 min to 90 %

282

and kept for 1 min. Finally, the starting conditions were re-adjusted in 1 min

283

and kept for 5.5 min.

284

The concentrations were calculated using response curves for each

285

analyte determined by analysis of defined amounts of labeled and unlabeled

286

nucleosides and nucleotide phosphates in different concentration ratios (from

287

0.003 to 6.72 mg/L; eight-point calibration) and organic acids (from 0.002 to

288

100 mg/L; eight-point calibration). Using the multiple reaction monitoring

289

(MRM) mode operating in the negative ionization mode (ESI-), nucleosides,

290

nucleotide phosphates and organic acids were analyzed in the effluent after

291

tuning the MS parameters for each compound. The following mass transitions

292

were used for the analytes and the corresponding internal standards: ATP

293

(m/z 506.1→158.9)/

294

15

N5-GTP (m/z 527.1→158.9), ADP (m/z 425.9→134.1) was quantitated using

295

15

N5-GMP (m/z 367.1→78.9), xanthosine (m/z 283.1→151.0), guanosine (m/z

15

N5-ATP (m/z 511.1→158.9), GTP (m/z 522.0→158.9)/


Page 12 of 43

Page 13 of 43


296

282.0→150.0), inosine (m/z 267.0→135.0), adenosine (m/z 266.1→134.0),

297

uridine (m/z 242.9→110.0), cytidin (m/z 242.1→109.0) and hypoxanthin (m/z

298

135.2→92.0) using

299

acid (m/z 167.2→124.1) was quantitated using

300

169.1→125.0) and succinic acid (m/z 117.0→98.9) was quantitated using 2H4-

301

succinic acid (m/z 121.1→77.0) as the internal standard. The Xevo TQ-S

302

mass spectrometer operated in negative electrospray (ESI-). The instrument

303

parameters were set as follows: capillary voltage (-2.2 kV), sampling cone (20

304

V), source offset (50 V), source temperature (150° C), desolvation

305

temperature (350° C), cone gas (150 L/h), desolvation gas (700 L/h), collision

306

gas flow (0.15 mL/min) and nebulizer gas (7 bar). Data was processed and

307

analyzed using MassLynx 4.1 and Targetlynx (Waters, Manchester, UK).

15

N5-AMP (m/z 351.0→78.9) as internal standard. Uric 15

N2-uric acid (m/z

308

For method validation, aliquots (400 µL) of artificial saliva (5000 mg/L

309

potassium bicarbonate, 640 mg/L potassium chloride, 150 mg/L sodium

310

carbonate, 206.2 mg/L α-amylase, 100 mg/L mucin in water) were spiked with

311

an aliquot (100 µL) of diluted stock solutions containing the target amino

312

acids, organic acids and nucleotides in triplicate and further processed as

313

described before. As control, an aliquot of the artificial saliva (400 µL) was

314

mixed with water (100 µL) instead of the target analytes and further processed

315

as mentioned above. The lower limit of quantitation (LLOQ) of each

316

compound was determined by dilution with water of the saliva samples until

317

the signal-to-noise (S/N) ratio of the analytes after quantitation was at least 9

318

(see Tables S2-5).

319

Comprehensive Gas Chromatography - Time of Flight - Mass

320

Spectrometry (GCxGC-ToF-MS). Aliquots (1.9 mL) of non-stimulated saliva



321

(t0) and saliva samples (t75) collected from four volunteers (2 male, 2 female)

322

stimulated with 6-gingerol were placed into sealed headspace vials (volume:

323

20 mL) at 37°C, spiked with an aliquot (0.1 mL) of a freshly prepared solution

324

of 2-furfurylthiol (50 mg/L) and, then, incubated at 37°C for up to 15 min. After

325

0, 5 and 15 min, respectively, an aliquot (3.0 mL) of a solution of 2-[α-2H2]-2-

326

furfurylthiol (1.35 mg/L) in a saturated CaCl2 solution was added and, after 15

327

min equilibration and 1:50 dilution with saturated CaCl2 solution, the volatiles

328

were analyzed by means of HS-SPME using a DVB/CAR/PDMS fiber (df

329

50/30 µm, 2 cm length; Supelco, Bellefonte, PA, USA) exposed to the

330

headspace at 40°C for 30 min using the Gerstel Agitator heating unit and

331

MPS autosampler. After thermal desorption of the fiber for 20 min at 250°C in

332

the split injector, analysis was performed on a Pegasus 4D GC×GC/TOF-MS

333

instrument (Leco, St. Joseph, MI, USA) consisting of an Agilent Technologies

334

GC model 7890A, a dual-stage quad-jet thermal modulator, and a secondary

335

oven coupled to a time-of-flight mass spectrometer providing unit mass

336

resolution. An Agilent split injector (Waldbronn, Germany) was used operated

337

by a Gerstel MPS autosampler (Mühlheim, Germany). Operated with helium

338

(2 mL/min) as the carrier gas, an Agilent DB-FFAP (30 m, 0.25 mm i.d.)

339

equipped with a deactivated pre-column (2 m, 0.53 mm, i.d.) was used in the

340

first dimension and operated from 40 °C (2 min) with 4 °C/min to 230 °C (4

341

min), while an Agilent VF-5 column (2 m, 0.15 mm i.d.) was used in the

342

second dimension using the following temperature program: starting at 70 °C

343

for 2 min, temperature was raised with 8 °C/min to 250 °C and, then, kept for

344

5 min prior to cooling. Mass spectra were acquired within m/z 40-250 at a rate


Page 14 of 43

Page 15 of 43


345

of 100 spectra/s. Data were elaborated using GC Image and GC Project

346

(Lincoln, Nebraska, USA).

347

In Vivo Breath Analysis using Proton Transfer Reaction Mass

348

Spectrometry (PTR-MS). In vivo analysis of 2-furfurylthiol in exhaled breath

349

as performed with a quadrupole PTR-MS (Ionicon Analytik GmbH, Innsbruck,

350

Austria). To select the major ions to be monitored during PTR-MS analysis, an

351

aqueous solution (100 mL) of 2-furfurylthiol (50 mg/L) was placed in a glass

352

flask (1000 mL) as reported earlier,41 headspace was drawn at 90 mL/min into

353

the PTR-MS and the mass intensities (mass range m/z 45-120) were

354

analyzed using the multiple ion mode (MID) with a dwell time of 100 ms per

355

mass. As 2-furfurylthiol mainly produced the fragment ions m/z 81 (100%) and

356

m/z 80 (20%), next to the molecular ion m/z 114 (30%), these ions were

357

selected for the in vivo analysis of 2-furfurylthiol.

358

For PTR-MS analysis, the panelists were asked to rinse the oral cavity

359

with water (8 mL) for 60 s and, then, to expectorate. In a “control” experiment,

360

the subjects took water (2 mL) as the stimulus-free vehicle into the mouth and

361

simulated chewing motions for 30 s. Thereafter, an aliquot (5 mL) of an

362

aqueous solution of 2-furfurylthiol (1.0 mg/L) was given to the panelists to

363

rinse the oral cavity for 30 s and, after expectorating the sample, 2-furfurylthiol

364

was measured directly by means of PTR-MS mouthspace analysis. A

365

comparative “stimulus” experiment was performed identical to the “control”

366

experiment by substituting the water vehicle (control) by an aliquot (2 mL) of

367

an aqueous solution of 6-gingerol (1.7 mmol/L). After measuring 20 cycles of

368

ambient air (background) by means of PTR-MS, the exhaled breath of the

369

volunteers was drawn at a rate of 90 mL/min through a heated transfer line



370

(120°C). The reaction chamber was held at 80°C and the drift voltage was set

371

at 600 V (2.10 mbar). After monitoring 60 cycles of 2-furfurylthiol in-vivo

372

breath release, again 20 cycles of ambient air were measured to get initial

373

conditions for the PTR-MS. The obtained data, expressed in counts per

374

second (cps) for each mass, were adjusted to a defined intensity of 107 cps

375

for the H3O+ ion. For practical reasons the H3O+ signal was detected at m/z 21

376

as H3O18+ with a constant isotope ratio of 1:500 relative to m/z 19. For visual

377

clarity, only the data of the most intense ion m/z 81 is displayed.

378

Sensory Studies. Intensity Rating of Salt Taste. In order to study the

379

influence of oral citric acid stimulation on the perceived saltiness of sodium

380

chloride solutions, the following sensory setup was used. The panelists were

381

asked to take water (4 mL) as the stimulus-free vehicle (control) into the oral

382

cavity, to perform chewing motions for 15 s, to expectorate and, then, to

383

evaluate the salt taste intensity of an aliquot (1 mL) of a NaCl solution (50

384

mmol/L) on a five-point scale from 0 (no impression) to 5 (very strong

385

impression). After a resting period of 1 min, the panelists were asked to take

386

an aliquot (4 mL) of the following stimuli solution into the mouth, to perform

387

chewing motions for 15 s and, then, to expectorate: stimuli used were water

388

adjusted to pH 2.2 and 6.8 with aqueous HCl, aqueous citric acid solutions

389

(5.2, 26.0, 78.0, and 156.0 mmol/L), aqueous ammonium citrate (156

390

mmol/L), aqueous malic acid (156 mmol/L), and an aqueous solution of citric

391

acid (156.0 mmol/L) and NaCl (70 mmol/L). Thereafter, oral cavity was

392

washed with water (15 mL) for 5 s and spitting out. After another cleansing

393

step (15 mL water, 5 s), the intensity of 1 mL of a NaCl (50 mM) solution was

394

rated again.


Page 16 of 43

Page 17 of 43


395

Intensity Rating of 2-Furfurylthiol. To rate the perceived after-smell

396

intensity after oral intervention with 2-furfurylthiol, panelists were asked to

397

clean the oral cavity for 60 s by washing with water (8 mL), then to take water

398

(2 mL) into the mouth while performing chewing motions for 30 s and, after

399

spitting out, an aliquot (5 mL) of a 2-furfurylthiol solution (20 µg/L) was taken

400

up while performing chewing motions for 15 s and, after expectoration, the

401

FFT intensity was evaluated on a scale from 0 (no impression) to 10 (very

402

strong impression). This procedure was repeated using 2 mL of a 6-gingerol

403

solution (1.7 mmol/L) instead of 2 mL of water.

404

Statistics. Data handling was done using Microsoft Excel 2016,

405

Perseus version 1.5.8.5 (Homepage, Paper) and GraphPad Prism versions

406

7.00

407

www.graphpad.comGraphpad Prism). The metabolome data (t15, t45 and t75) of

408

all stimuli-activated saliva samples were normalized to the corresponding non-

409

stimulated control sample (t0). To evaluate significant differences between the

410

perceived intensity of 2-furfurylthiol and NaCl, respectively, before and after

411

stimulation with 6-gingerol and citric acid, the Wilcoxon signed rank test was

412

performed. All P < 0.05 were considered as significant different, P < 0.001

413

was marked with three stars and P < 0.0001 was marked with four stars.

for

Windows

(GraphPad

Software,

La

Jolla

California

USA,

414 415 416

RESULTS AND DISCUSSION

417 418

Very recently, tryptic digestion of saliva samples collected after stimulation

419

with taste and trigeminal stimuli, respectively, followed by nano-HPLC-MS/MS



420

and label-free protein quantitation mapped out chemosensory stimulus-

421

specific changes in the composition of the saliva proteome.30 Re-investigation

422

of the proteome data collected,30 revealed an increase of the abundance of

423

salivary isoform 1 of the sulfhydryl oxidase 1 upon oral stimulation with 6-

424

gingerol, e.g. the abundance of this enzyme increased by factors of 8 and 16

425

from non-stimulated saliva (t0) to 15 (t15) and 75 s after stimulation (t75),

426

respectively (Figure 1). As sulfhydryl oxidases are known to catalyze the

427

oxidation of mercaptans,31-33 the following studies were performed to answer

428

the question as to whether an increase in salivary sulfhydryl oxidase 1

429

translates into an altered sensory perception of odor-active mercaptans.

430

Effect on 6-Gingerol Stimulation on Perception of 2-Furfurylthiol.

431

Non-stimulated, resting saliva (t0) and the post-stimulus saliva sample t75,

432

collected 75 s after oral 6-gingerol challenge and showing the highest

433

abundance of salivary isoform 1 of sulfhydryl oxidase 1 (Figure 1), were

434

collected and incubated in vitro with 2-furfurylthiol (FFT), a roasty and sulfury

435

smelling key food odorant identified in thermally processed foods, such as,

436

e.g. roasted coffee, roasted sesame seeds, and thermally treated meat,

437

respectively.42 After 0, 5 and 15 min, respectively, enzymatic reactions were

438

terminated by adding a saturated CaCl2 solution and, after adding 2-[α-2H2]-2-

439

furfurylthiol as the internal standard and equilibration, the FFT concentration

440

was determined by means of stable isotope dilution analysis (SIDA) using

441

headspace solid phase extraction (SPME), followed by GCxGC-ToF-MS. In

442

the presence of non-stimulated saliva (t0), FFT degraded to give 77 and 65 %

443

of the initial FFT concentration 5 and 15 min after incubation, respectively

444

(Figure 2A). In comparison, post-stimulus saliva (t75) induced a significantly


Page 18 of 43

Page 19 of 43


445

accelerated FFT decline, e.g. only 55 and 41 % of the odorant were recovered

446

after 5 and 15 min, respectively (Figure 2A), thus being well in line with the

447

increased abundance of sulfhydryl oxidase 1 in this saliva sample.

448

As furfuryl disulfide was expected as the main reaction product formed

449

by sulfhydryl oxidase 1 catalyzed oxidation of FFT, the rise of furfuryl disulfide

450

was quantitively monitored in the same sample set. The non-stimulated saliva

451

samples (t0) convertedd 21% of the initial FFT concentration to form 2-furfuryl

452

disulfide after 5 min and 27% of the FFT after 15 min of incubation (Figure

453

2B). The increased abundance of the sulfhydryl oxidase 1 in the saliva sample

454

t75 lead to the formation of furfuryl disulfide out of 78% and 92% of the FFT

455

after 5 min and 15 min of incubation (Figure 2B), respectively, which clearly

456

demonstrates a faster conversion of FFT to furfuryl disulfide in saliva samples

457

with elevated levels of sulfhydryl oxidase 1.

458

To verify these in vitro data by means of in in mouth experiment, four

459

healthy volunteers were asked to take water (control) or an aqueous solution

460

of 6-gingerol (1.7 mmol/L; stimulus experiment), respectively, into the oral

461

cavity, to simulate chewing motions for 30 s and, then, to expectorate.

462

Thereafter, an aqueous solution containing 2-furfurylthiol (5.0 µg) was given to

463

the panelists to rinse the oral cavity for 30 s and, after expectorating the

464

sample, 2-furfurylthiol was measured directly (0 min) and after 2 min,

465

respectively, by means of PTR-MS breath analysis monitoring the most

466

intense fragment ion m/z 81 (Figure 3). Although, individual differences were

467

found in the levels of exhaled FFT, orosensory stimulation with 6-gingerol

468

induces a drastic decrease in exhaled FFT independent on the subject (Figure

469

3).



470

To answer the question as to whether the reduced levels of FFT in the

471

subjects‘ breath translate also into a decreased after-smell perception of the

472

thiol, the panelists repeated the up-mentioned control and stimulus

473

experiment of the PTR-MS study with an aqueous FFT solution, containing 20

474

µg/L of that mercaptan as typically found in a roast coffee brew,34 and then,

475

were asked to rate the perceived after-smell intensity on a 10-point intensity

476

scale immediately (0 min) and 2 min after expectorating the FFT solution,

477

respectively (Figure 4). Right upon the FTT challenge (0 min), the perceived

478

odor intensity was rated with a score of 3.9 (control) and 1.5 (after

479

stimulation). After 2 min, FFT could be still perceived with an intensity of 2.0 in

480

the control experiment, whereas the 6-gingerol stimulation (intensity 0.3)

481

almost diminished the perception of the odorant in the after-breath (Figure 4).

482

Taking all these data into consideration, it may be concluded that

483

orosensory pre-stimulation with 6-gingerol induced an increased abundance

484

of the salivary enzyme sulfhydryl oxidase 1 which oxidizes mercaptans like

485

the sulfury-roasty smelling 2-furfurylthiol to lower its levels in the exhaled

486

breath and, in consequence, the perceived sulfury after-smell. The

487

chemosensory modulation of sulfhydryl oxidase 1, therefore, may be one

488

component of an efficient molecular network triggering oral cleansing

489

mechanisms after food ingestion and may open new avenues for innovative

490

oral care applications suppressing a long-lasting after-smell. As the stimulus-

491

induced salivary proteome response was observed instantaneously upon

492

stimulation, the observed modulation of the sulfhydryl oxidase 1 activity in

493

saliva is very likely to result from the release of the enzyme from preformed

494

vesicles and not from de novo synthesis which in exocrine cells were shown


Page 20 of 43

Page 21 of 43


495

to take about 30 min to pass from the rough endoplasmatic reticulum to the

496

condensing vacuoles.43

497

Effect of Orosensory Stimulation on Salivary Metabolome. To

498

study the impact of oro-sensory stimulation on the salivary metabolome, first,

499

resting saliva (t0) was collected, volunteers were then asked to take an aliquot

500

of water (control) or an aqueous stimulus solution of citric acid (S1),

501

aspartame (S2), iso-α-acids (S3), mono sodium L-glutamate (S4), sodium

502

chloride (S5), 6-gingerol (S6), hydroxy-α-sanshool (S7), and hydroxy-β-

503

sanshool (S8), respectively, in the mouth. After performing chewing motions

504

for 15 s, the subjects were asked to expectorate to deliver the corresponding

505

stimulus sample (t15), followed by saliva collections after 45 and 75 s (post-

506

stimulus samples t45 and t75), respectively. Minerals, organic acids, amino

507

acids, nucleotides and nucleosides were then quantitatively determined in

508

each saliva sample and, after normalizing the data obtained upon (t15) and

509

after stimulation (t45, t75) to the corresponding control sample (t0), the entire

510

set of quantitative data was heat-mapped in Figure 5.

511

Overall, the highest impact of all taste stimuli was observed after

512

stimulation with citric acid (Figure 5A). Although, the stimulation with citric acid

513

has been reported to induce a significant saliva flow increase,28,30,38,44-46 the

514

concentrations of minerals increased immediately upon stimulation (t15) and

515

even persisted on elevated levels after stimulation (t45, t75) compared to the

516

unstimulated saliva (t0), e.g. the sodium levels increased from 1.8 (t0) to 21.6

517

mmol/L 45 s after stimulation. Similarly, the oral stimulation with 6-gingerol,

518

which is also known to enhance saliva flow,28,30 revealed an increase of

519

salivary sodium concentrations from 2.0 (t0) to 11.3 mmol/L (t45) (Figure 5B).



520

On the basis of these data, it may be concluded that massive stimulation of

521

the saliva flow leads to increased mineral concentration in saliva, most likely

522

because the sodium ions cannot be completely re-absorbed in the striated

523

ducts of the salivary glands.1,47 Interestingly, the other saliva components

524

analyzed showed only minor changes upon chemosensory stimulation and

525

slightly decreased in the saliva samples after stimulation (t15-t75) compared to

526

the corresponding control samples (t0) (Figure 5), thus implying a dilution of

527

the metabolites in saliva due to an increased saliva flow.

528

To investigate the dose-dependent impact of citric acid on the salivary

529

secretion of anions and cations, aqueous solutions of citric acid in increasing

530

concentrations of 5.2, 26.0, 78.0 and 156.0 mmol/L, respectively, were used

531

for orosensory intervention and both, anions and cations were quantitated in

532

saliva (Figure 6). Whereas the diluted citric acid solution (5.2 mmol/L) induced

533

only marginal changes in the salivary levels of anions and cations, increasing

534

concentrations of the stimulus showed a huge impact, e.g. the 26 mmol/L

535

citric acid solution revealed an increase of sodium, potassium, phosphate and

536

chloride ions from 1.96, 4.71, 0.14, and 9.92 mmol/L prior to stimulation (t0) to

537

8.05, 8.57, 3.68, and 14.86 mmol/L, respectively, right upon stimulation (t15).

538

At 78 mmol/L, citric acid stimulation revealed an increase of the salivary

539

sodium levels from 1.8 mmol/L (t0) over 9.6 mmol/L (t15) to reach a maximum

540

of 12.43 mmol/L after 45 s, followed by a decrease to 5.55 mmol/L (t75). Also

541

phosphate was largely increased from 0.15 (t0) to the maximum of 5.36

542

mmol/L (t15) which confirms literature findings that sour stimuli induce

543

phosphate ion secretion to neutralize the acids and to diminish the drop of the

544

pH of the oral cavity.1,48 When the oral stimulation was done with a 156


Page 22 of 43

Page 23 of 43


545

mmol/L citric acid solution, the sodium concentration increased 11 times from

546

unstimulated saliva (t0) to reach a maximum of 21.6 mmol/L after 45 s. In

547

comparison, only a 4-fold increase was found for chloride ions, e.g.

548

concentrations raised from 12.1 (t0) to 39.3 mmol/L (t45).

549

As the perceived intensity of a salty taste has been attributed to the

550

sensory contrast,49,50 that means the difference between basal NaCl levels in

551

saliva and the concentration of NaCl in a stimulus, the question arose as to

552

whether the increase in salivary NaCl levels after citric acid stimulation may

553

translate in an altered salt taste perception. Therefore, a sensory study was

554

performed and the panelists asked to rate the perceived intensity of a sodium

555

chloride solution before and after stimulation with individual stimuli solutions.

556

After rinsing the oral cavity with water, the panelists were asked to take up

557

water (control) and to perform chewing motions for 15 s, to expectorate and,

558

then, to rate the salty taste of an aqueous NaCl solution (50 mmol/L) on a five-

559

point intensity scale. After a resting period of 60 s, a stimulus experiment was

560

performed by substituting the water blank with an aqueous stimulus solution,

561

followed by two water rinse phases prior to sensory evaluation of the NaCl

562

solution (50 mmol/L) (Figure 7A).

563

After stimulation with low levels of 5.2 and 26.0 mmol/L citric acid,

564

which has shown not or only slightly to affect saliva’s mineral composition

565

(Figure 6A, B), the perceived saltiness was slightly increased or unaffected

566

when compared to the control sample (w/o) (Figure 7B). Increasing the citric

567

acid levels to 78 and 156 mmol/L, leading to higher salivary mineral levels

568

(Figure 6), induced a significant decline of the perceived saltiness of the NaCl

569

solution, e.g. the salt intensity dropped from 1.5 to 0.9 and 0.5, respectively



570

(Figure 7B). An additional experiment showed that after stimulation with 156

571

mmol/L citric acid, no significant differences were found between a 50 mmol/L

572

(control) and a 70 mmol/L NaCl solution (Figure 7C). These data clearly

573

indicate that the higher basal levels of salivary sodium after citric acid

574

stimulation decreases the concentration difference to the test stimulus of NaCl

575

(50 mmol/L) which, due to a decreased sensory contrast,49,50 is then

576

interpreted as being less salty.

577

Another experiment was performed with two water samples adjusted to

578

pH 6.8 and 2.2 (matching the pH of the 156 mmol/L citric acid solution) as

579

stimuli in order to investigate as to whether the concentrations of free

580

hydroxonium ions are responsible for the effects observed. Interestingly, the

581

salty taste intensity increased from 1.5 to 2.0 and 2.1, respectively (Figure

582

7D), thus demonstrating that the organic acids rather than the free protons

583

play a role in salty taste modulation.

584

In order to answer the question as to whether the protonated citric acid

585

or the citrate anion is inducing the modulation of salt taste perception, another

586

experiment was performed using ammonium citrate (156 mmol/L, pH 6.9) as

587

the stimulus (Figure 7E). In comparison to the decrease of the perceived salt

588

taste intensity from 1.5 to 0.5 when citric acid (156 mmol/L) was used as

589

stimulus (Figure 7B), the ammonium citrate stimulation did not effect the

590

perceived saltiness of the 50 mmol/L NaCl solution (Figure 7E). In contrast,

591

stimulation with malic acid (156 mmol/L) again significantly decreased the

592

perceived saltiness of the NaCl test solution from 1.5 to 0.6 (Figure 7F).

593

Therefore, it may be concluded that neither free protons, nor the carboxylate


Page 24 of 43

Page 25 of 43


594

anions, but the protonated organic acids lower the perceived saltiness of NaCl

595

solutions by lifting the basal sodium concentrations in saliva.

596

Taking all data into account, it may be concluded that oro-sensory

597

stimulation with taste and trigeminal compounds triggers changes in the

598

composition of the salivary proteome and metabolome which translate into a

599

functional modulation of odor and taste perception. Orosensory pre-

600

stimulation with 6-gingerol was found to induce an increased abundance of

601

the salivary sulfhydryl oxidase 1 catalyzing the oxidative decline of free

602

mercaptans, such as, e.g. the sulfury-roasty smelling 2-furfurylthiol, thus

603

leading to lower odorant levels in the exhaled breath and, in consequence, a

604

reduction of the perceived sulfury after-smell. The chemosensory modulation

605

of sulfhydryl oxidase 1 may be, therefore, considered as an important

606

component of an efficient molecular network triggering oral cleansing

607

mechanisms after food ingestion and may open new avenues for innovative

608

oral care applications suppressing a long-lasting after-smell. Moreover, oro-

609

sensory stimulation with citric acid was found to induce a strong increase of

610

concentrations of minerals and, in particular, sodium ions, while the other

611

salivary metabolites were rather unaffected. Due to the elevated basal levels

612

of salivary sodium after citric acid stimulation, NaCl test stimuli were perceived

613

as significantly less salty, most likely due to a decreased sensory contrast.49,50

614

Although the exact mechanisms that fine-tune odor and taste sensitivity are

615

yet poorly defined, these data indicate the modulation of the salivary proteome

616

and metabolome to be a major peri-receptor event in the oral cavity and may

617

play an important role in odor and taste recognition. Utilizing the knowledge



Page 26 of 43

618

gap on such peri-receptor events reveals a competitive advantage in the

619

development of premium flavors or flavors with oral health benefits.

620 621 622

ACKNOWLEDGEMENT

623

We thank Ines Otte and Sami Kaviani-Nejad, Leibniz-Institute for Food

624

Systems Biology at the Technical University of Munich, for acquisition of

625

GCxGC-ToF-MS/MS data.

626 627

SUPPORTING INFORMATION AVAILABLE

628

Table

629

http://pubs.acs.org.

S1-S5

are

available

free

of

charge

630

631


via

the

Internet

at

Page 27 of 43


632

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Mese, H.; Matsuo, R. Salivary secretion, taste and hyposalivation. J. Oral Rehab. 2007, 34, 711-723.

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Kaufmann, E.; Lamster, I. B. The diagnostic applications of saliva - a review. Crit. Rev. Oral Biol. Med. 2002, 13, 197–212.

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Humphrey, S. P.; Williamson, R. T. A review of saliva: Normal

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Chauncey, H. H.; Shannon, I. L. Glandular mechanisms regulating the

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electrolyte composition of human parotid saliva. Ann. N. Y. Acad. Sci.

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Lendenmann, U.; Grogan, J.; Oppenheimer, F. G. Saliva and dental

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dimensional electrophoresis of human salivary proteins from patients

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with sialoadenopathy. Arch. Oral Biol. 1993, 38, 1135–1139.

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Identification of protein components in human acquired enamel pellicle

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and whole saliva using novel proteomics approaches. J. Biol. Chem.

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2003, 278, 5300–5308.

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(12) Ghafouri, B.; Tagesson, C.; Lindahl, M. Mapping of proteins in human

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saliva using two-dimensional gel electrophoresis and peptide mass

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fingerprinting. Proteomics 2003, 3, 1003–1015.

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FIGURE LEGENDS

Figure 1.

Time course of the abundance of sulhydryl oxidase 1 (QSOX1, isoform 1) after stimulation with 6-gingerol (1.7 mmol/L). Saliva samples were collected before stimulation (t0), after a 15 s stimulation with 6-gingerol (t15), and 30 (t45) and 60 s after stimulation (t75). The thin grew lines indicate the individual replicates, the dark bold line indicates the mean value. Protein data were extracted from our recently published proteome analysis.30

Figure 2.

Time course of (A) the decline of 2-furfurylthiol (FFT) and (B) the generation of furfuryl disulfide after incubation of resting saliva (t0; blue line) and gingerol-stimulated saliva (t75; red line).

Figure 3.

Breath analysis of four individuals (A-D) monitoring the fragment ion m/z 81 of FFT by means of PTR-MS prior to (blue labelled) and after stimulation (red labelled) using 6-gingerol (1.7 mmol/L).

Figure 4.

Odor intensity perceived directly (0 min) or 2 min after rinsing the mouth with an aqueous solution of 2-furfurylthiol (5 mL; 20 µg/L) prior to stimulation (blue columns) and after stimulation with 6gingerol (red columns), respectively.

Figure 5.

Heatmap of

quantitative metabolome data measured upon

stimulation (t15) and 30 (t45) or 60 s (t75) after stimulation with citric acid (A), 6-gingerol (B), hydroxy-α-sanhool (C), hydroxy-β-sanshool (D), iso-α-acids (E), monosodium L-glutamate (F), NaCl (G) and aspartame (H). Data are normalized to the corresponding control sample (t0). Figure 6.

Influence of the concentration of aqueous citric acid solutions used for orosensory stimulation on the minerals in saliva; (A) 5.2 mmol/L, (B) 26 mmol/L, (C) 78 mmol/L, (D) 156 mmol/L. Data are given as the mean values of eight panelists.


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Figure 7.

(A) Experimental setup for the sensory evaluation of an aqueous NaCl solution (50 mmol/L) prior to stimulation (control) and after orosensory

intervention

with

different

stimuli

(stimulation

experiment). (B) Perceived intensity of the NaCl solution (50 mmol/L) prior to (w/o; black bar) and after pre-stimulation with (B) citric acid solutions of various concentrations (5.2 – 156.0 mmol/). (C) Perceived intensity of a NaCl solution (50 mmol/L) prior to (w/o; black bar) stimulation and a NaCl solution (70 mmol/L) after prestimulation with a citric acid solution (156 mmol/L). Perceived intensity of a NaCl solution (50 mmol/L) after stimulation with (D) water adjusted to pH 2.2 and 6.8, respectively, (E) ammonium citrate (156 mmol/L), or malic acid (156 mM).



Figure 1 (Bader et al.)


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