Discovery of a Thiamine-Derived Taste Enhancer in Process Flavors

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Chemistry and Biology of Aroma and Taste

Discovery of a Thiamine-Derived Taste Enhancer in Process Flavors Laura Brehm, Manon Jünger, Oliver Frank, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01832 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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Journal of Agricultural and Food Chemistry

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Discovery of a Thiamine-Derived Taste Enhancer

2

in Process Flavors

3 4

Laura Brehm1, Manon Jünger1, Oliver Frank1 and Thomas Hofmann1,2,3*

5 6

1Chair

7

München, Lise-Meitner-Str. 34, D-85354 Freising, Germany, and 2Bavarian Center for

8

Biomolecular Mass Spectrometry, Gregor-Mendel-Straße 4, D-85354 Freising,

9

Germany. 3 Leibniz-Institute for Food Systems Biology, Technical University of Munich,

10

of Food Chemistry and Molecular and Sensory Science, Technische Universität

Lise-Meitner-Strasse 34, D-85354 Freising, Germany

11 12 13 14 15 16 17

*

18

PHONE

+49-8161/71-2902

19

FAX

+49-8161/71-2949

20

E-MAIL

[email protected]

To whom correspondence should be addressed

21 22

ACS Paragon Plus Environment


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ABSTRACT

25 26

Targeted quantitation of 48 basic taste-active compounds in commercial meat-like

27

process flavors, calculation of dose-over-threshold factors, and basic taste re-

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engineering, followed by activity-guided fractionation revealed, next to L-glutamate and

29

5’-ribonucleotides, a series of N-acetylated amino acids and S-((4-amino-2-

30

methylpyrimidin-5-yl) methyl)-L-cysteine as taste modulating compounds. The N-

31

acetylated amino acids imparted kokumi enhancement with rather high taste

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thresholds ranging up to 1800 µmol/L (N-acetyl methionine) in model broth. In

33

comparison, S-((4-amino-2-methylpyrimidin-5-yl) methyl)-L-cysteine, found to be

34

formed by a Maillard-type reaction of thiamine and cysteine, is reported for the first

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time to exhibit strong kokumi enhancement above a low threshold concentration of 120

36

µmol/L (model broth). These results will open new avenues towards a knowledge-

37

based optimization of thiamine-containing process flavors.

38 39

40

KEYWORDS: sensomics, kokumi, process flavors, Maillard reaction, thiamine, taste

41

modulation


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INTRODUCTION

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In the western world the number of vegetarians and vegans is increasing over time.1

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Thus, the market for vegetarian and vegan substitution products is growing.2 To fulfill

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the request of the consumers for vegetarian and vegan foods, so-called process flavors

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are used to deliver the attractive flavor profile of thermally treated chicken, beef, and

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pork, respectively, without using any animal-derived ingredients.3,4 The International

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Organization of the Flavor Industry (IOFI) defines process flavors as a thermal process

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flavoring prepared for its flavoring properties by heating food ingredients and/or

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ingredients permitted for use in foodstuffs or in process flavorings.5,6 In process flavor

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manufacturing,

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ribonucleotides, phospholipids, yeast extracts, and hydrolyzed vegetable protein,

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respectively, are thermally treated under controlled conditions (temperature, reaction

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time, pH).5

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components like amino acids and peptides, the formation of Strecker aldehydes and

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acids from amino acids, as well as the formation of meaty odorants from thiamine are

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considered the most important reactions leading to the generation of process flavors.4,5

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Although the consumer’s acceptance of thermally processed foods is also

61

influenced by non-volatile taste-active compounds, flavor research has been mainly

62

focused just on the volatile aroma fraction.7 However, the umami and kokumi tasting

63

substances also play an important role for the savory and meaty taste impression of

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process flavors. Besides L-glutamate and ribonucleotides, several taste modulating

65

molecules have been found to be generated by Maillard-type reactions,8–12 e.g. the

66

sweet and umami enhancing alapyridaine from alanine and hexoses,8 the umami

67

enhancing

68

ribonucletide 5’-GMP and reducing carbohydrates,9 and the kokumi enhancing N-(1-

precursors

like

amino

acids,

reducing

sugars,

thiamine,

The Maillard reaction between reducing carbohydrates and amino

N2-(1-carboxyethyl)

guanosine

monophosphate


from

the

yeast’s


69

methyl-4-oxoimidazolidin-2-ylidene) α-amino acids from creatinine and reducing

70

carbohydrates.10 Several studies investigated the taste activity of thermally processed

71

mixtures of peptides and carbohydrates, e.g. the molecular weight fraction of 1 to 5 kDa

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isolated from a reaction mixture of hydrolyzed soybean protein or sunflower protein,

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respectively, with carbohydrates were reported to significantly increase the kokumi

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activity, mouthfullness and continuity of savory broth solutions.11,12

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In recent years, the so-called sensomics approach and using the taste dilution

76

analysis (TDA) and the comparative taste dilution analysis (cTDA), respectively, to

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locate and identify the key molecules imparting the typical taste of foods and model

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reaction systems, revealed previously unknown taste modulators in chicken broth,

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cooked crustaceans, thermally processed avocado, stewed beef meat/vegetable

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broth, and golden chanterelles.9,10,13–18

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Although there is a strongly growing market for meaty process flavors, the

82

knowledge on the chemical structure and formation mechanisms of taste enhancing

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molecules generated from natural materials during process flavor manufacturing is still

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rather fragmentary. Any knowledge of such previously unknown taste enhancers would

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help to optimize the recipe and the manufacturing parameters targeting premium

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process flavors with meat-like authenticity. The objective of present study was to

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apply the sensomics approach for the first time on commercial process flavors in order

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to locate, identify, and sensorially characterize the key taste and taste modulating

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principles.

90 91 92 93

MATERIALS AND METHODS

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Chemicals. The following compounds were obtained commercially: acetonitrile,

96

methanol (J.T. Baker, Netherlands), ethyl acetate (VWR, AnalaR Normapur, France),

97

formic acid, (Merck, Darmstadt, Germany). Solvents used for LC-MS/MS analysis were

98

of LC-MS grade (Honeywell, Seelze, Germany). Water for HPLC separation was

99

purified using a Milli-Q water advantage A10 water system (Millipore, Schwalbach,

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Germany). For sensory analysis, bottled water (Evian, Danone, Wiesbaden, Germany)

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was used. Yeast extract (Gistex XII LS) was obtained from Food Ingredients

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Distribution (Werne, Germany).

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nucleosides, nucleotides, carbohydrates, and the N-acetyl amino acids N-acetyl

104

alanine (1), N-acetyl isoleucine (2), N-acetyl leucine (3), N-acetyl methionine (4), N-

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acetyl phenylalanine (5), and N-acetyl tyrosine (6) were obtained from Sigma Aldrich

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(Steinheim, Germany). Stable isotope labeled amino acids, organic acids, nucleotides,

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and nucleosides were purchased from Cambridge Isotope Laboraties Inc (Tewksbury,

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MA, USA). N-((4-Amino-2-methylpyrimidin-5-yl) methyl) formamide (7) was purchased

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from Carbosynth (Berkshire, United Kingdom). Deuterated solvents were obtained

110

from

111

monophosphate, N2-(ß-D-glucosyl) guanosine monophosphate, and the labeled

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standard N2-([13C3]-1-Carboxyethyl)-guanosin-5’-monophosphat were synthesized as

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reported recently.9 Eight commercially available meat-like process flavors, three of

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them made without using yeast extract (PF1-PF3) and four of them containing yeast

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extract (PF4-PF8), wherein PF1-PF7 contained thiamine as ingredient were obtained

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from the food industry. For the sensory detection of taste modulating compounds, a

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model broth was prepared by dissolving sodium chloride (2.9 g), monosodium L-

118

glutamate (1.9 g), maltodextrin (6.4 g), and yeast extract (2.1 g) in water (1 L).

Sigma

Aldrich

L-amino

(Steinheim,

acids, organic acids, inorganic acids,

Germany).

N2-(1-Carboxyethyl)

guanosine

119

Quantitative Analysis of Basic Taste Compounds. For quantitation of the

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basic tastants, a defined amount (1 mg) of the process flavor (PF1 - PF8) was ACS Paragon Plus Environment


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dissolved in water (1 mL) and, after membrane filtration (0.45 µm), the sample was

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analyzed directly or after 1:5 dilution with water prior to analysis. The quantitation of

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cations, anions and carbohydrates was performed by means of high-performance ion

124

chromatography (HPIC)16 and the free amino acids,14,16,19,20 nucleotides and

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nucleosides,13 N-modified nucleotides,9 organic acids,18,20 and γ-glutamyl peptides19

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were quantitated by means of LC-MS/MS following the procedures reported earlier.

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Sequential Solvent Fractionation of Process Flavor PF5. A portion (30 g) of

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PF5 was dissolved in water (200 mL), extracted with distilled ethyl acetate (2 x 200

129

mL), and the combined organic layers were separated from solvent in vacuum to obtain

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the ethyl acetate extractables. The residual aqueous layer was freeze-dried, an aliquot

131

of the lyophilizate (2.5 g) was dissolved in water (10 mL) and extracted with acetonitrile

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(20 mL). After phase separation, the upper acetonitrile phase was decanted and the

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water phase was extracted with acetonitrile (4 times, 20 mL each). The acetonitrile

134

(ACN) phases were combined, the solvent was removed in vacuum, and the residue

135

as well as the remaining water phase were lyophilized to afford the ACN extract and

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the water solubles. The extracts were stored at -20 °C until further use.

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Size Exclusion Chromatography (SEC) of the ACN-Extract. An aliquot of the

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lyophilized ACN-extract (500 mg) was dissolved in water (5 mL) and applied onto a

139

glass column packed with LH-15 material (GE, Healthcare, Uppsala, Sweden). The

140

compounds were eluted with water adjusted to pH 4.0 (with formic acid) and with a

141

flow rate of 3 mL/min. The effluent was detected at λ=200 nm by means of an UV

142

detector (UV-2075, Jasco, Groß-Umstadt, Germany) and separated into eight fractions

143

(fractions SEC-I – SEC-VIII). To collect the fractions a CF-2 Fraction Collector

144

(Spectrum Europe B.V., Breda, Netherlands) was used. The combined fractions were

145

lyophilized and stored at −20 °C prior to sensory experiments and chemical analysis.


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Subfractionation of SEC-Fraction III. Aliquots of the lyophilized SEC

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fraction III (250 mg) were dissolved in water (10 mL). After membrane filtration

148

(0.45 μm) the solution was fractionated on a 250 x 10 mm i.d.,10 µm, Nucleodur-C18-

149

Pyramid column (Machery Nagel, Dueren, Germany). Monitoring the effluent with an

150

UV-detector (λ=254 nm), chromatography was performed at a flow rate of 4.5 mL/min

151

using aqueous formic acid (0.1%) as solvent A and acetonitrile as solvent B. Starting

152

with 0% B for 5 min, the gradient was increased to 60% B in 22 min. Within another 2

153

min the gradient was increased to 100% B and maintained for 1 min. Afterwards, the

154

column was conditioned to 0% B in 2 min and held for 3 min for equilibration. The

155

HPLC effluent was collected separately into eleven fractions, namely SEC-III-1 to SEC-

156

III-11, lyophilized and stored at −20 °C prior to sensory experiments and chemical

157

analysis.

158

Identification of Taste-Modulating Compounds in Subfraction III-1. The

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lyophilized HPLC fraction III-1 (20 mg) was taken up in water (2 mL), membrane filtered

160

(0.45 µm) and, then, separated by means of semipreparative hydrophilic interaction

161

liquid chromatography (HILIC-HPLC) on a 250 × 21.5 mm i.d., 10 µm, TSKgel Amide-

162

80 column (Tosoh Bioscience, Stuttgart, Germany). Monitoring the effluent with an UV-

163

detector (λ=254 nm), chromatography was performed at a flow rate of 8 mL/min using

164

aqueous formic acid (0.1%) as solvent A and acetonitrile as solvent B. Starting with

165

80% B for 3 min, the gradient was decreased to 0% B in 9 min and held for 1 min.

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Within another 2 min, the gradient was increased to 80% B and held for 3 min for

167

equilibration. The HPLC effluent was collected separately to give two subfractions,

168

namely III-1-a and III-1-b, which were lyophilized twice. UV-Vis, LC-MS/MS, TOF-MS

169

and 1D/2D-NMR experiments revealed the chemical structure of the target compounds

170

in fraction SEC-III-1-a as N-((4-amino-2-methylpyrimidin-5-yl) methyl) formamide (7)

171

and in fraction III-1-b as S-((4-amino-2-methylpyrimidin-5-yl) methyl)-L-cysteine (8). ACS Paragon Plus Environment


172

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N-((4-Amino-2-methylpyrimidin-5-yl) methyl) formamide (7), Figure 6: UV-Vis 1H

173

λmax = 254 nm (MeOH/H2O, 50/50, v/v); UPLC-TOF-MS: m/z 167.1061

174

(400 MHz, MeOD-d4, 298 K): δ (ppm) 8.15 [d, J = 0.8 Hz, 1H, H-C(1’)], 7.94 [s, 1H, H-

175

-C(6)], 4.25 [s, 2H, H-C(7)], 2.39 [s, 3H, H-C(8)]. 13C NMR (100 MHz, MeOD-d4, 298 K):

176

δ (ppm) 167.84 [C(4)], 164.47 [C(1’)], 163.48 [C(2)], 155.35 [C(6)], 112.35 [C(5)] , 36.45

177

[C(7)], 24.85 [C(8)].

178

NMR

S-((4-Amino-2-methylpyrimidin-5-yl) methyl)-L-cysteine 8, Figure 6: UV-Vis m/z 243.0915; 1H NMR

179

λmax = 254 nm (MeOH/H2O, 50/50, v/v); UPLC-TOF-MS:

180

(400 MHz, MeOD-d4, 298 K): δ (ppm) 8.07 [s, 1H, H-C(6)], 3.96 [t, J = 5.3 Hz, 1H; H-

181

C(2‘)], 3.77 [d, J = 15.0 Hz, 1H¸ Ha-C(7)], 3.70 [d, J = 15.0 Hz, 1H, Hb-C(7)], 3.00 [d, J

182

= 5.3 Hz, 2H, H-C(3‘)], 2.57 [s, 3H, H-C(8)];

183

δ (ppm) 173.49 [C(1‘)], 164.31 [C(4)], 162.58 [C(2)], 142.55 [C(6)], 112.23 [C(5)], 54.13

184

[C(2‘)], 31.95 [C(3‘)], 28.78 [C(7)], 21.63 [C(8)].

13C

NMR (100 MHz, MeOD-d4, 298 K):

185

Thermal Reaction of Thiamine and Cysteine. A binary solution of thiamine (1

186

mmol) and cysteine (1 mmol) in aqueous phosphate buffer (10 mL; pH 7; 0.1 mol/L)

187

was thermally treated at 120°C for 120 min. After cooling, the mixture was separated

188

by RP-HPLC on a 250 x 10.00 mm Phenyl-Hexyl column (Phenomenex

189

Aschaffenburg) into nine fractions, amongst which fraction 2 contained the target

190

molecule exhibiting a pseudomolecular ion with m/z 243.0915. Comparison of the

191

spectroscopic (1H NMR, LC-MS) and chromatographic data demonstrated the identity

192

of the reaction product obtained from fraction 2 and the taste modulator 8 isolated from

193

process flavor PF5, thus confirming S-((4-amino-2-methylpyrimidin-5-yl)methyl)-L-

194

cysteine to be generated from thiamine and cysteine.

195

Sensory Analyses. Sensory Panel Training. Eight female and nine male

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panellists (23-40 years in age), who had no history of known taste disorders and who

197

had given the informed consent to participate in the present sensory tests, were trained ACS Paragon Plus Environment

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in weekly training sessions for at least two years in order to became familiar with the

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taste language and methodologies used, to evaluate the taste of aqueous reference

200

solutions (2.0 mL; pH 5.9): sucrose (50 mmol/L) for sweet taste,

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(20 mmol/L) for sour taste, NaCl (20 mmol/L) for salty taste, caffeine (1 mmol/L) for

202

bitter taste, monosodium L-glutamate (3 mmol/L) for umami taste. To train the activity

203

of mouthfullness enhancement and complexity increase, coined kokumi activity, the

204

panelists were requested to compare the gustatory impact of a blank model broth

205

(control) with a solution of reduced glutathione (5 mmol/L) in model both.21,22

L-lactic

acid

206

Taste Profile Analysis. Portions (5.0 g) of process flavors were dissolved in

207

water (1 L) and model broth (1 L), respectively, and the pH value was adjusted to 5.4

208

with trace amounts of 1% formic acid. The panelists were asked to rate the intensity of

209

the taste qualities umami, saltiness, bitterness, sweetness, sourness as well as kokumi

210

sensation on a scale from 0 (not perceivable) to 5 (strongly perceivable). When the

211

process flavors were dissolved in model broth, the rating was compared to a model

212

broth without any additives. The ratings of the individual panelists were averaged.

213

Comparative Taste Dilution Analysis. To locate the umami and kokumi tasting

214

substances, a comparative taste dilution analysis was performed according to the

215

literature.8 The lyophilized SEC and HPLC fractions, respectively, were dissolved with

216

the same volume of model broth and the pH-value was adjusted to 5.4 with trace

217

amounts of formic acid (1% in water). The stock solutions were diluted successively

218

1:1 with model broth until a dilution factor of 256 was reached and, then, the solutions

219

were presented in order of increasing concentrations to the sensory panel using a duo-

220

trio test setup with model broth as reference and blank (control). The comparative taste

221

dilution (cTD) factor is defined as the first dilution step where a difference between the

222

sample and the blank were detectable. The cTD-factors of all panelists were averaged.



223

Taste Threshold Determination. A solution of the test compound in a defined

224

concentration was prepared in water (intrinsic taste) and in model broth (taste

225

enhancement), respectively, then successively diluted 1:1 and presented to the

226

panelists in order of increasing concentrations using a duo-trio test procedure with

227

water or model broth as reference and blank (control). The first dilution step where a

228

difference between the blank and the sample is detectable is defined as the taste

229

threshold. The taste threshold was calculated as the geometric mean of the individual

230

data.

231

High-Performance Liquid Chromatography (HPLC). The HPLC apparatus

232

(Jasco, Gross-Umstadt, Germany) used consisted of a binary high-pressure HPLC

233

pump system PU-2080 Plus, an AS-2055 Plus autosampler, a DG-2080-53 degasser,

234

a MD-2010 Plus type diode array detector (Jasco, Gross-Umstadt, Germany), and a

235

Rh 7725i type Rheodyne injection valve (Rheodyne, Bensheim, Germany). Data

236

acquisition was done by means of Chrompass Chromatography Data System, Version

237

1.9 (Jasco, Gross-Umstadt, Germany).

238

Liquid Chromatography-Mass spectrometry (LC-MS). LC-MS/MS analysis

239

was performed either using a Dionex Ultimate 3000 HPLC system connected to API

240

4000 QTrap MS/MS device, or a Shimadzu Nexera X2 system (Shimadzu, Duisburg,

241

Germany) connected to a 5500 QTrap MS/MS system (AB Sciex, Darmstadt,

242

Germany). Running in the positive or negative electrospray ionization (ESI+, ESI-)

243

mode, both instrument setups were operated using the following conditions: zero grade

244

air served as nebulizer gas (55 psi) and turbo gas for solvent drying (65 psi, 450 °C);

245

nitrogen served both as curtain gas (35 psi) and collision gas (8.7× 10−7 psi);

246

dissociation potential (-2 V) and entrance potential (-10 V). Both quadrupoles were set

247

at unit resolution. ESI+ and ESI− mass and product ion spectra were acquired with

248

direct flow infusion. For ESI+, the ion spray voltage was set at +5500 V and −4500 V ACS Paragon Plus Environment

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for ESI−. Energies for declustering potential (DP), entrance potential (EP), collision

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energy (CE), and cell exit potential (CXP) as well as MS/MS parameters for measuring

251

in the MRM mode were optimized for each compound individually to detect the

252

fragmentation of molecular ions into specific product ions after collision with nitrogen.

253

For instrumental control and data acquisition, Sciex Analyst software v1.6 was used.

254

UPLC/Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). An aliquot (0.1 mg

255

- 1mg) of the sample, dissolved in methanol/water (30/70, v/v; 1mL), was injected into

256

an Acquity UPLC core system (Waters UK Ltd., Manchester, UK) connected to a

257

SYNAPT G2 HDMS spectrometer (Waters UK Ltd., Manchester, UK) operating in a

258

positive electrospray (ESI) modus with the following parameters: capillary voltage

259

(+2.0 kV), sampling cone (20 V), source temperature (120 °C), desolvation

260

temperature

261

Chromatographic separations were performed on a column (2.1 x 150 mm, 1.7 µm,

262

BEH C18, Waters UK Ltd., Manchester, UK) operated at 45 °C with a solvent gradient

263

(flow rate 0.4 mL/min) of 0.1% aqueous formic acid (solvent A) and 0.1% formic acid

264

in acetonitrile (solvent B). Starting with 5% B the ratio was increased in 4 min to

265

100% B. The instrument was calibrated over a mass range from m/z 100 to 1200 using

266

a solution of sodium formate (0.5 mmol/L) in 2-propanol/water (9/1, v/v). All data were

267

lock mass corrected using leucine enkephaline as the reference (m/z 556.2771 for

268

[M+H]+; m/z 554.2615 for [M-H]-). Data acquisition and analysis were done by using

269

the MassLynx software (version 4.1; Waters).

(450 °C),

cone

gas

(5 L/h),

and

desolvation

gas

(850 L/h).

270

Nuclear Magnetic Resonance Spectroscopy (NMR). 1D- and 2D-NMR

271

experiments were performed on a Bruker 400 MHz Avance III spectrometer (Bruker,

272

Rheinstetten, Germany) equipped with a Z-gradient 5 mm multinuclear observe probe

273

(BBFOplus). MeOD-d4 (600 µL) was used as solvent and chemical shifts are reported

274

in parts per million referenced to the MeOD-d4 solvent signals. Data processing was ACS Paragon Plus Environment


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performed by using Topspin NMR software (version 3.2; Bruker, Rheinstetten,

276

Germany) and MestReNova 10.0 (Mestrelab Research, Santiago de Compostela,

277

Spain). For quantitative NMR spectroscopy (qNMR), the spectrometer was calibrated

278

by using the ERETIC 2 tool using the PULCON methodology as reported earlier.23 The

279

isolated signal at 3.96 ppm (t, J = 5.3 Hz, 1H) was used for absolute quantitation of 8

280

using a defined sample of L-tyrosine as the external standard and its specific

281

resonance signal at 7.10 ppm (m, 2H) for analyses.

282 283

RESULTS AND DISCUSSION

284 285

Aimed at identifying the key molecules contributing to the typical savory taste profile of

286

process flavors, eight commercial samples, three of them made without using yeast

287

extract (PF1-PF3) and four of them containing yeast extract (PF4-PF8), were used for

288

sensory analysis in water to evaluate the intrinsic taste profiles and in model broth to

289

evaluate their taste modulating activities, respectively.16 Independent on the process

290

flavor, the umami taste impression was by far most pronounced. The yeast-free

291

process flavors PF1 – PF 3 showed a moderate umami taste intensity with PF2

292

exhibiting the highest umami (1.6) and kokumi activity (0.6) and PF1 the lowest umami

293

(1.1) and kokumi (0.4) intensity (Figure 1, A). In comparison, the yeast-containing

294

samples PF4 - PF8 showed a stronger umami taste (2.0 - 2.3) with the exception of

295

sample PF7 judged with a low intrinsic umami intensity of 1.1 (Figure 1, B).

296

Sensory evaluation of the process flavors in model broth revealed a moderate

297

umami enhancing activity for the samples PF1 - PF3 when compared to the blank

298

model broth (Figure 1, C). Interestingly, the yeast containing process flavors (PF4 -

299

PF8) showed a strong umami enhancement (2.23.0) when added to the model broth

300

(Figure 1, D). In addition, these samples also increased the kokumi activity of the


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model broth (+0.7). Among all samples tested, PF5 and PF6 showed the highest

302

enhancement effect of the umami (3.4 and 3.1) and the kokumi activity (1.3) in model

303

broth. In the following, literature-known basic taste compounds were quantitated in the

304

process flavors to evaluate their contribution to the taste activity of the process flavors.

305

Quantitation of Basic Tastants in Process Flavors. Amino acids, organic

306

acids, γ-glutamyl peptides, nucleotides and N-modified nucleotides were quantitated

307

by means of SIDA-LC-MS/MS and carbohydrates, cations, and anions were

308

determined by means of HPIC.13,17,19,20,24–26 Thereafter, the dose-over-threshold (DoT)

309

factor was calculated as the ratio of the concentration and the taste recognition

310

threshold of each individual compound.27 As nucleotides like 5’-GMP and 5’-IMP show

311

strong umami taste enhancement, their DoT-factors were calculated by using their

312

threshold concentration determined in an aqueous monosodium L-glutamate solution

313

(3 mmol/L) instead of water.28

314

The 48 basic taste compounds classified into six groups (A-F) depending on

315

their taste quality (Figure 2, A). Due to small DoT-factors (0.1), most of the

316

quantitated substances did not affect the taste activity of the process flavors (Figure

317

2, B). The DoT-factors of L-glutamic acid varied within the PFs samples from

Discovery of a Thiamine-Derived Taste Enhancer in Process Flavors

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