New Taste-Active 3-(O-Î²-d-Glucosyl)-2-oxoindole ... - ACS Publications

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Chemistry and Biology of Aroma and Taste

New Taste-Active 3-(O-#-D-Glucosyl)-2-oxoindole-3-acetic Acids and Diarylheptanoids in Cimiciato-Infected Hazelnuts Barbara Singldinger, Andreas Dunkel, Dominic Bahmann, Claudia Bahmann, Daniel Kadow, Bernward Bisping, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01216 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Journal of Agricultural and Food Chemistry

1

New Taste-Active 3-(O-β-D-Glucosyl)-2-oxoindole-3-acetic

2

Acids and Diarylheptanoids in Cimiciato-Infected Hazelnuts

3 4

Barbara Singldinger1, Andreas Dunkel1,2, Dominic Bahmann3, Claudia

5

Bahmann3, Daniel Kadow4, Bernward Bisping3 and Thomas Hofmann1,2,5*

6 1

7

Chair of Food Chemistry and Molecular and Sensory Science, Technische

8

Universität München, Lise-Meitner-Str. 34, D-85354 Freising, Germany, 2

9

Bavarian Center for Biomolecular Mass Spectrometry,Technical University of

10

Munich, Gregor-Mendel-Straße 4, D-85354 Freising, Germany,

11

3

12

of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, D-22609 Hamburg, Germany,

Food Microbiology and Biotechnology, Hamburg School of Food Science, University 4

13 5

14

August Storck KG, R&D Chocolates, Waldstr. 27, D-13403 Berlin, Germany,

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

15

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

16 17 18

*

19

PHONE

+49-8161/71-2902

20

FAX

+49-8161/71-2949

21

E-MAIL

[email protected]

To whom correspondence should be addressed

22

ACS Paragon Plus Environment

1


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ABSTRACT

24 25

Activity-guided fractionation in combination with sensory analytics, LC-TOF-MS, and

26

1D/2D-NMR

27

diarylheptanoids asadanin, giffonin P and the previously not reported (E)-7,9,10,13-

28

tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one

29

oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-heptaen as well as the yet

30

unknown astringent compounds 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3-O-6´-

31

galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate and 3-(O-β-D-glycosyl) dioxindole-3-

32

acetic acid in Cimiciato-infected hazelnuts exhibiting a bitter off-taste. Quantitative

33

LC-MS/MS studies, followed by dose/activity considerations confirmed for the first

34

time asadanin to be the key contributor to the bitter taste of Cimiciato-infected

35

hazelnuts. Furthermore, quantitative studies demonstrated that neither the physical

36

damage alone, nor a general microbial infection is able to initiate a stress-induced

37

asadanin generation, but most likely either specific Cimiciato-specific microorganisms

38

associated with the bugs, or specific chemical stimulants in the bugs’ saliva is the

39

cause triggering asadanin biosynthesis. Finally, also germination was found for the

40

first time to activate diarylheptanoid biosynthesis, resulting in higher contents of bitter

41

tasting phytochemicals and development of the bitter off-taste.

spectroscopy

enabled

the

identification

of

and

the

bitter

tasting

4,12,16-trihydroxy-2-

42 43

KEYWORDS:

taste,

bitter,

hazelnuts,

44

diarylheptanoids, asadanin, giffonin P

Corylus

avellana

L.,

Cimiciato,

45 46


2

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47

INTRODUCTION

48 49

Because of its pleasant aroma and attractive taste profile, hazelnuts (Corylus

50

avellana L.) are used as key ingredients in confectionary, chocolate, and snack

51

products. With a volume of about 580,000 metric tons per year (~85% of world

52

market), Turkey is by far the largest producer of hazelnuts, followed by Italy with an

53

annual production volume of about 90,000 metric tons. But plant growing conditions

54

and the hazelnuts harvest procedure differ largely between both countries of origin.1

55

While hazelnut harvest in Turkey is performed manually by more than 320,000

56

farmers and dried under the sun without moisture control, hazelnuts in Italy are

57

harvested mechanically by a small number of highly professionalized farming

58

operations, followed by highly controlled drying and processing methods, resulting in

59

a better quality of hazelnuts.2,3

60

Hazelnuts, in particular when originating from Turkey, have been reported to

61

develop a sporadic bitter off-taste upon storage that is maintained throughout

62

roasting to exhibit a flavor defect in final products and leads to consumer complaints,

63

resulting in a serious problem for the hazelnut producers and the manufacturing

64

industry.4,5 Just very recently, by means of a sensomics approach,6–15 the so-called

65

asadanin, 1 (Figure 1), which has been discovered in wood extracts of Ostrya

66

japonica in 1968,16 has been identified as a main contributor to the bitter off-taste of

67

hazelnuts.17 Asadanin belongs to the group of cyclic diarylheptanoids which consist

68

of a basic biphenyl (biphenyl type) or a meta, para ether-bridged biphenyl (diphenyl

69

ether type) connected via a C7-alkyl chain and were reported as phytochemicals in

70

the bark, leaves and branches of various plant families, such as, e.g. Aceraceae,

71

Betulaceae,

Burseraceae,

72

Myricaceae,

and

Casuarinaceae,

Zingiberaceae.18–23

Next

Juglandaceae, to


asadanin

Leguminosae, (1),

additional 3


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73

diarylheptanoids, coined giffonins A-P, were identified in the leaves and the bark of

74

hazelnuts,24,25 however, their bitter taste impact remains elusive.

75

A decrease in flavor quality accompanied by an increase in bitter taste and

76

tissue necrosis has also been reported in raw hazelnuts upon infection by bugs,

77

belonging to the hemipteran like Gonocerus acuteangulatus and Coreus marginatus,

78

respectively.26–28 Depending on the maturity states of the hazelnut fruit, the attack of

79

the bug can induce different types of damages to the nut, e.g. the so-called

80

“Cimiciato” that occurs if the bugs attack the hazelnut kernel in an advanced stage of

81

maturity. One hypothesis is that upon infection, the hemipteran releases saliva

82

containing enzymes, such as, e.g. proteases, amylases, esterase, and lipases,

83

inducing a biotic stress response and change in metabolism of the kernel.29–32

84

As additional diarylheptanoids may add to the bitter off-taste contribution of

85

asadanin (1), in particular after Cimiciato infection, the objective of the present study

86

was to apply a sensomics approach to map the bitter tasting compounds in

87

Cimiciato-infected hazelnuts, to determine the chemical structures and human

88

recognition threshold concentrations of the major taste contributors by means of LC-

89

TOF-MS and 1D/2D-NMR spectroscopy, and to quantitate the target compounds in

90

non-infected premium vs. infected hazelnut samples. As also germination processes

91

are well-known to alter metabolism of the hazelnut kernel,33–35 additional quantitative

92

studies were performed to study whether germination-induced metabolic changes

93

may be considered as an additional factor driving biosynthesis of asadanin and other

94

bitter compounds.

95 96 97


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MATERIALS AND METHODS

99 100

Chemicals.

The

following

compounds

were

obtained

commercially:

101

acetonitrile, methanol (J.T. Baker, Netherlands), ethyl acetate, n-pentane (VWR

102

prolabo chemicals, AnalaR Normapur, France), formic acid, (Merck, Darmstadt,

103

Germany); Solvents used for HPLC-MS/MS analysis were of LC-MS grade

104

(Honeywell, Seelze, Germany), n-pentane and ethyl acetate were distilled before

105

using, all other solvents were of HPLC grade (Merck Darmstadt, Germany).

106

Deuterated solvents (DMSO-d6, methanol-d4) were obtained from Sigma Aldrich (St.

107

Louis, USA). L-Tyrosine for qNMR was purchased from Sigma Aldrich (Fluka

108

Analytical, Steinheim, Germany). The internal standards (+/-)-myricanol was received

109

from Extrasynthese (Genay, France), L-tryptophan-d5 from Cambridge Isotope

110

Laboratories Inc. (Andover, USA). Water for HPLC separation was purified by the

111

use of a Milli-Q water advantage A 10 water system (Millipore, Molsheim, France).

112

For sensory analysis, bottled water (Evian, Danone Waters Deutschland, Frankfurt

113

am Main, Germany) was used. Reference material of asadanin (1) was purified as

114

reported recently.17

115

Premium hazelnuts (PN) were hand-selected by experts from the hazelnut

116

manufacturing industry. Cimiciato-infected hazelnut kernels (CN) from Turkey were

117

provided by the German food industry after sorting by a visual inspection by a trained

118

expert

119

microorganisms (bacteria, molds, yeast) were collected in the black sea region

120

cultivation area in Turkey by an experienced microbiologist of the Biocenter Klein-

121

Flottbek (Hamburg, Germany). The hazelnut mark was provided by the German food

122

industry and consisted of roasted and highly ground hazelnuts.

panel.

Hazelnut

samples

used

for

identification


and

culturing

of

5


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123

Sequential Solvent Extraction of Cimiciato-Infected Hazelnuts (CN). A

124

portion (300 g) of Cimiciato-infected powdered hazelnuts, obtained by grinding deep-

125

frozen kernels using a GM 300 type mill (Retsch, Haan, Germany) at 4000 rpm for

126

40 s, was extracted three times with methanol/water (70/30, v/v; 1 L) at room

127

temperature. The extracts were combined, methanol removed in vacuum at 39 °C,

128

and the aqueous solution was then either freeze-dried to obtain extract CN, which

129

was taken up in 0.3% ethanolic water for sensory studies (spiking experiment), or

130

sequentially extracted with n-pentane (4 x 0.5 L), followed by ethyl acetate (3 x

131

0.8 L). The corresponding organic extracts were separated from solvent in vacuum at

132

39 °C, followed by lyophilization to obtain the pentane solubles (fraction I), the ethyl

133

acetate extractables (fraction II), and the water solubles (fraction III), respectively.

134

The residual hazelnut material was freeze-dried twice to result in the insoluble

135

fraction IV, which did not show any taste activity. The lyophilized fractions I-III were

136

dissolved in 3% ethanolic water and sensorially evaluated by means of a

137

comparative taste profile analysis (Table 1).

138

Sequential Solvent Extraction of Premium Hazelnut Kernels (PN). A portion

139

(300 g) of premium raw hazelnuts, obtained by grinding deep-frozen kernels using a

140

GM 300 type mill (Retsch, Haan, Germany) at 4000 rpm for 40 s, was extracted three

141

times with methanol/water (70/30, v/v; 1 L) at room temperature. The extracts were

142

combined, methanol removed in vacuum at 39 °C, followed by lyophilization to obtain

143

the methanol/water extractables (PN extrac), which was taken up in 0.3% ethanolic

144

water for a sensory spiking experiment.

145

Separation of Fraction II by Means of Medium Pressure Liquid

146

Chromatography (MPLC). An aliquot (350 mg) of lyophilized Cimiciato-fraction II

147

was dissolved in acetonitrile/water (12/88, v/v; 3.5 mL) and separated by MPLC on a

148

150 x 40 mm i.d. polypropylene cartridge filled with 25-40 µm LiChroprep RP-18 ACS Paragon Plus Environment

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149

material (Merck, Darmstadt, Germany). The MPLC apparatus (Büchi, Flawil, Swiss)

150

consisted of a binary pump module C-605, a control unit C-620, a fraction collector

151

C-660, and a Sedex LT-ELSD detector Model 80 (Sedere, Alfortville, France). MPLC

152

was performed at a flow rate of 40 mL/min to give 12 fractions, namely II-1 to II-12.

153

Using 0.1% formic acid in water (v/v) as solvent A and acetonitrile as solvent B,

154

chromatography was performed with the effluent monitored using a Sedex LT-ELSD

155

detector and the following gradient: 3 min at 0% B, within 2 min to 5% B, held 1 min

156

at 5% B, within 2 min to 10% B, increased in 8 min to 15% B, held 5 min at 15% B,

157

increased in 7 min to 20% B, within 10 min to 25% B, held 3 min at 25% B, increased

158

in 6 min to 30% B, maintained 5 min at 30% B, within 8 min to 100% B, held 10 min

159

at 100% B, decreased in 7 min to 0% B and, finally, held for 10 min at 0% B. Each of

160

the 12 fractions from Cimiciato-infected hazelnut kernels, collected by means of a C-

161

660 type fraction collector, was separated from solvent in vacuum at 39 °C and, after

162

taking up the residues in water and freeze-drying twice, the fractions were kept at

163

minus 20°C until used for the taste dilution analysis (TDA) or further fractionation,

164

respectively.

165

UHPLC-TOF-MS Analysis of MPLC-Fractions II-1 to II-12. Aliquots (1 mg) of

166

lyophilized fractions II-1 to II-12 were dissolved in acetonitrile/water (70/30, v/v, 1 mL)

167

and injected into an Acquity UPLC core system (Waters, Manchester, UK) connected

168

to a Synapt G2 HDMS spectrometer (Waters). Chromatographic separations were

169

performed on a 2.1 x 150 mm, 1.7 µm, BEH C18 column (Waters) operated at 45°C

170

with a solvent gradient (flow rate 0.4 mL/min) of 0.1% aqueous formic acid (solvent

171

A) and 0.1% formic acid in acetonitrile (solvent B): 0 min, 5% B, in 4 min to 100% B.

172

On comparison of chromatographic (retention time) and spectroscopic data (1H NMR,

173

LC-MS/MS) with those of the purified reference compound,17 asadanin (1) was

174

identified in fraction II-8, evaluated with the highest bitter impact (Figure 2). ACS Paragon Plus Environment

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Isolation of 3-(O-β-D-Glycosyl)-2-oxoindole-3-acetic Acids 2 and 3 from

176

Fraction II-7 and II-4. The taste-active fraction II-7 contained, next to some amounts

177

of the bitter tasting asadanin (1), an unknown astringent compound (2) showing

178

m/z 541.2 as the pseudomolecular ion (ESI-). To isolate this compound, fraction II-7

179

was dissolved in acetonitrile/water (12/88, v/v; 22.9 mg/mL) and then separated by

180

means of preparative RP-HPLC on a 250 × 21 mm i.d., 5 µm, Nucleodur Pyramid

181

C18 column (Macherey-Nagel, Düren, Germany). Using a flow rate of 20 mL/min with

182

0.1% formic acid in water (v/v) as solvent A and acetonitrile as solvent B,

183

chromatographic separation was executed with the effluent monitored at 254 nm:

184

starting with a mixture 5% B and 95% A, held at 5% B for 3 min, increased to 25% B

185

in 4 min, held isocratically with 25% B for 13 min, increasing the acetonitrile content

186

to 30% B over 2 min, held at 30% B for 2 min, decreased in 5 min to 5% B and finally

187

held at 5% B for 6 min. A total of 19 subfractions were collected, namely fractions II-

188

7-1 to II-7-19, which were separated from solvent in vacuum at 39 °C, freeze-dried

189

twice, and the residue of subfraction II-7-9, containing the unknown target compound

190

2, analyzed by means of UV-Vis, LC-MS/MS, TOF-MS, and 1D/2D-NMR. The

191

astringent compound 2 was identified as the previously unknown 2-(3-hydroxy-2-

192

oxoindolin-3-yl) acetic acid 3-O-6´-galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate.

193

Fraction II-4, containing an astringent compound (3) with m/z 368.1 as the

194

pseudomolecular ion (ESI-), was dissolved in acetonitrile/water (10/90, v/v;

195

22.1 mg/mL) and, after membrane filtration, separated by preparative RP-HPLC on a

196

250 × 21 mm i.d., 5 µm, Nucleodur Pyramid C18 column (Macherey-Nagel). Using a

197

flow rate of 20 mL/min with 0.1% formic acid in water (v/v) as solvent A and

198

acetonitrile as solvent B, chromatography was performed with the effluent monitored

199

at 254 nm: starting with a mixture 5% B and 95% A, held at 5% B for 3 min,

200

increasing the acetonitrile content to 10% B over 5 min, increase to 20% B within ACS Paragon Plus Environment

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12 min, held isocratically with 20% B for 3 min, decrease in 2 min to 5% B and finally

202

held at 5% B for 6 min. The effluent was separated to give 19 subfractions, namely II-

203

4-1 to II-4-19. The individually collected fractions were freed from solvent in vacuum

204

at 39 °C, freeze-dried twice, and the residue of fraction II-4-4, containing compound

205

3, was used for structural analysis. UV-Vis, LC-MS/MS, LC-TOF-MS, and 1D/2D-

206

NMR led to the unequivocal identification of the target compound 3 as 3-(-O-β-D-

207

glycosyl)dioxindole-3-acetic acid, that has been previously reported from orange peel

208

and suggested to be a growth regulator.36

209

2-(3-Hydroxy-2-oxoindolin-3-yl)

acetic

acid

3-O-6´-galactopyranosyl-2“-

210

(2“oxoindolin-3“yl) acetate, 2, Figure 1: LC-MS (ESI-): m/z 541.2 [M-H]-; LC-MS/MS

211

(DP = -110 V): m/z 541.2, 189.9; LC-TOF-MS (ESI+): m/z 565.2 [M+Na]+; LC-TOF-

212

MS (ESI-): m/z 541.1470 [M-H]- (measured), m/z 541.1458 (calcd. for [C26H25N2O11]-);

213

1

214

3,00 [1H, m, H-C(8β)], 3,15 [1H, m, H-C(8α)], 3.06 [m, 1H, H-C(3´)]*, 3.19 [m, 1H, H-

215

C(5´)], 3.20 [m, 1H, H-C(4´)], 3.21 [m, 1H, H-C(2´)], 3.79 [m, 1H, H-C(3´´)], 4.20-4,02

216

[1H, m, H-C(6´β)], 4,25 [1H, m, H-C(6´α)], 4,32 [1H, d, J = 7,7 Hz, H-C(1´)], 6.84 [m,

217

1H, H-C(7´´)], 6.9 [m, 1H, H-C(7)], 6.94 [m, 1H, H-C(5)], 7.01 [m, 1H, H-C(5´´)], 7.21

218

[m, 2H, H-C(6)/H-C(6´´)], 7.3 [dd, 1H, J=13.6, 7.52 Hz, H-C(4´´)], 7.44 [dd, 1H,

219

J=13.5, 7.56 Hz, H-C(4)];

220

[C(3´´)], 44.7 [C(8)], 64.8 [C(6´)], 71.2 [C(5´)], 74.8 [C(4´)], 75.0 [C(3´)], 77.7 [C(2´)],

221

80.7 [C(3)], 100.1 [C(1´´)], 110.9 [C(7´´)], 111.1 [C(7)], 123.0 [C(5)], 123.5 [C(5´´)],

222

125.2 [C(4´´)], 127.1 [C(4)], 129.3 [C(6´)], 130.4 [C(6)], 130.9 [C(4a´´)], 131.0 [C(4a)],

223

143.6 [C(7a´´)], 172.4 [C(9´´)], 175.1 [C(9)], 179.2 [C(2)], 181.3 [C(2´´)].

H NMR (500 MHz; MeOD-d4): δ 2.85 [m, 1H, H-C(8´´β)], 3,11 [1H, m, H-C(8´´α)],

13

C NMR (126 MHz; MeOD-d4): δ 35.5 [C(8´´)], 43.4

224

3-(-O-β-D-Glycosyl)dioxindole-3-acetic acid, 3, Figure 1: LC-MS (ESI-): m/z

225

368.1 [M-H]-; LC-MS/MS (DP = -20 V): m/z 368.0, 144.1; LC-MS-TOF (ESI+): m/z

226

392.1 [M+Na]+, LC-TOF-MS (ESI-): m/z 368.0998 [M-H]- (measured), m/z 368.0982 ACS Paragon Plus Environment

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227

(calcd. for [C16H18NO9]-); 1H NMR (400 MHz; MeOD-d4) δ 2.92 [m, 1H, H-C(5´)], 3,2

228

[m, 2H, H-C(2´)/H-C(3´)], 3,25 [m, 1H, H-C(4´)], 3,22 [1H, m, H-C(8β)], 3,34 [1H, m,

229

H-C(8α)], 3,58 [1H, dd, J = 12,01, 5,58 Hz, H-C(6´β)], 3,71 [1H, dd, J = 12,09,

230

2,06 Hz, H-C(6´α)], 4.18 [d, 1H, J=5.22 Hz, H-C(1´)], 6.9 [d, 1H, J=7.54 Hz, H-C(7)],

231

7.04 [dd, 1H, J=8.67, 7.54 Hz, H-C(5)], 7.3 [dd, 1H, J=8.65, 7.54 Hz, H-C(6)], 7.55 [d,

232

1H, J=7.54 Hz, H-C(4)];

233

71,3 [C(4´)], 74.8 [C(3´)], 77.7 [C(5´)], 77.8 [C(2´)], 80,7 [C(3)], 100.5 [C(1´)], 111.4

234

[C(7)], 123.2 [C(5)], 127.4 [C(4)], 127.7 [C(4a)], 131.4 [C(6)], 144.6 [C(7a)], 172.3

235

[C(9)], 178.8 [C(2)].

13

C NMR (100 MHz; MeOD-d4): δ 42.2 [C(8)], 62.5 [C(6)],

236

Isolation of Diarylheptanoids 4 and 5 from Fraction II-6. Fraction II-6 was

237

dissolved in acetonitrile/water (20/80, v/v; 15.5 mg/mL) and, after membrane

238

filtration, separated by preparative RP-HPLC on a 250 × 21 mm i.d., 5 µm, Nucleodur

239

Pyramid C18 column (Macherey-Nagel). Using 0.1% formic acid in water (v/v) as

240

solvent A and acetonitrile as solvent B and a flow rate of 20 mL/min, chromatography

241

was performed with the effluent monitored at 254 nm: starting with a mixture of 20%

242

B and 80% A, increasing the acetonitrile content to 40% B over 13 min, decreasing in

243

2 min to 20% B and finally held at 25% B for 5 min. The effluent was separated into

244

nine subfractions, namely II-6-1 to II-6-9, which were separated from solvent in

245

vacuum at 39 °C, freeze-dried twice, and the residues obtained were used for the

246

sensorial and structural analysis. Fractions II-6-5 and II-6-6, both showing bitter taste

247

activity, were analyzed by means of UV-Vis, LC-MS/MS, TOF-MS and 1D/2D-NMR to

248

assign

249

tricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexaene

250

giffonin P in hazelnut leaves,25 and the previously unknown (E)-7,9,10,13-

251

tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one (5).

the

taste

active

phytochemicals

as

3,8,9,10,11,12,17-heptahydroxy-


(4),

also

known

as

10

Page 11 of 56

252


3,8,9,10,11,12,17-Heptahydroxy-tricyclo[12.3.1.12,6]nonadeca

253

1(18),2,4,6(19),14,16-hexaene (giffonin P), 4, Figure 1: LC-MS (ESI-): m/z 361.1 [M-

254

H]-; LC-MS/MS (DP = -5 V): m/z 361.1, 241.0; LC-TOF-MS (ESI+): m/z 385.1

255

[M+Na]+, LC-TOF-MS (ESI-) : m/z 361.1327 [M-H]- (measured), m/z 361.1287 (calcd.

256

for [C19H21O7]-); 1H NMR (500 MHz; MeOD-d4): δ 3.02 [m, 2H, H-C(13)], 2.90-3.06

257

[m, 2H, H-C(7)], 4.01 [d, 1H, J=9.87 Hz, H-C(11)], 4.07 [s, 1H, H-C(9)], 4.18 [d, 1H,

258

J=9.92, 3.93 Hz, H-C(10)], 4.26 [dd, 1H, J=9.25, 5.89 Hz, H-C(12)], 4.74 [dd, 1H,

259

J=11.43, 3.78 Hz, H-C(8)], 6.74 [d, 1H, J=1.75 Hz, H-C(18)], 6.77 [d, 1H, J=2.07 Hz,

260

H-C(19)], 6.81 [s, 1H, H-C(16)], 6.82 [s, 1H, H-C(4)], 7.03-7.07 [m, 2H, H-C(5), H-

261

C(15)];

262

[C(12)], 70.1 [C(9)], 70.3 [C(8)], 79.5 [C(10)], 117.11 [C(6)], 117.13 [C(4)], 127.4

263

[C(1), C(2)], 130.2 [C(15)], 130.3 [C(5)], 130.4 [C(6)], 130.5 [C(14)], 135.2 [C(19)],

264

135.4 [C(18)], 152.8 [C(17)], 152.8 [C(3)].

265

13

C NMR (125 MHz; MeOD-d4): δ 35.1 [C(7)], 36.7 [C(13)], 69.1 [C(11)], 70.0

(E)-7,9,10,13-Tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-en-11-one,

5,

266

Figure 1: LC-MS (ESI-): m/z 359.1 [M-H]-; LC-MS/MS (DP = -95 V): m/z 359.0,

267

238.9; LC-TOF-MS (ESI+): m/z 383.1 [M+Na]+; LC-TOF-MS (ESI-): m/z 359.1137

268

(measured), m/z 359.1131 (calcd. for [C19H19O7]-); 1H NMR (500 MHz; DMSO-d6): δ

269

2,66 [2H, dd, J = 16,82, 7,7 Hz, H-C(12β), H-(8β)], 2,87 [2H, dd, J = 16,80, 4,62 Hz,

270

H-C(12α), H-C(8α)], 3.61 [dd, 2H, J=7.32, 4.84 Hz, H-C(13), H-C(7)], 6.80 [d, 2H,

271

J=7.85 Hz, H-C(2), H-C(16)], 6.91 [t, 2H, J=7.53, H-C(4), H-C(18)], 7.15 [t, 2H,

272

J=7.74, H-C(3), H-C(17)], 7.22 [d, 2H, J=7.34 Hz, H-C(5), H-C(19)];

273

(125 MHz; DMSO-d6): δ 34.2 [C(8), C(12)], 41.9 [C(7), C(13)], 109.1 [C(2), C(16)],

274

121.1 [C(4), C(18)], 123.6 [C(5), C(19)], 127.6 [C(3), C(17)], 129.5 [C(6), C(14)],

275

142.8 [C(1), C(15)], 172.2 [C(10)], 178.2 [C(11), C(9)].

13

C NMR

276

Isolation of Diarylheptanoids in Fraction II-10. Fraction II-10 was dissolved in

277

acetonitrile/water (60/40, v/v; 15.0 mg/mL) and, after membrane filtration, separated ACS Paragon Plus Environment

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by preparative RP-HPLC on a 250 × 21 mm i.d., 5 µm, Nucleodur Pyramid C18

279

column (Macherey-Nagel). Using a flow rate of 20 mL/min with 0.1% formic acid in

280

water (v/v) as solvent A and acetonitrile as solvent B, chromatography was

281

performed with the effluent monitored at 254 nm: starting with a mixture 20% B and

282

80% A, held at 20% B for 3 min, increasing to 100% B over 27 min, decreasing in

283

3 min to 20% B and finally held at 20% B for 5 min. The effluent was separated into

284

subfractions II-10-1 and II-10-2, which were freed from solvent in vacuum at 39 °C,

285

freeze-dried twice, and used for structural and taste analysis. By means of UV-Vis,

286

LC-MS/MS, TOF-MS and 1D/2D-NMR, the structure of the key bitter compound in

287

fraction II-10-1 was determined to be the previously unknown 4,12,16-trihydroxy-2-

288

oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-heptaene (6), Figure 1.

289

4,12,16-Trihydroxy-2-oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-

290

heptaene, 6, Figure 1: LC-MS (ESI-): m/z 311.1 [M-H]-; LC-MS/MS (DP = -55 V): m/z

291

311.1, 161.1; LC-TOF-MS (ESI+): m/z 313.1 [M+H]+; LC-TOF-MS (ESI-): m/z

292

311.1310 [M-H]- (measured), m/z 311.1283 (calcd. for [C19H19O4]-);

293

(500 MHz; MeOD-d4): δ 1.29 [m, 2H, H-C(10)], 1.91 [m, 2H, H-C(12)], 2.09 [m, 2H, H-

294

C(9)], 2.78 [m, 2H, H-C(13)], 3.38 [m, 1H, H-C(11)], 5.53 [m, 1H, H-C(8)], 6.30 [d, 1H,

295

J=11.35 Hz, H-C(7)], 6.53 [d, 1H, J=1.43 Hz, H-C(19)], 6.59 [dd, 1H, J=8.34, 1.61 Hz,

296

H-C(5)], 6.80-6.82 [m, 3H, H-C(4), H-C(16), H-C(17)], 6.91 [s, 1H, H-C(18)]; 13C NMR

297

(125 MHz; MeOD-d4): δ 27.6 [C(9)], 28.9 [C(13)], 36,3 [C(12)], 37.5 [C(10)], 74.2

298

[C(11)], 116.4 [C(19)], 117.2 [C(4), C(16)], 120.9 [C(17)], 123.3 [C(5)], 124.6 [C(18)],

299

130.2 [C(14)], 130.5 [C(6)], 131.1 [C(7)], 131.2 [C(8)], 146.1 [C(3)], 148.6 [C(2)],

300

148.9 [C(1)], 153.5 [C(15)].

1

H NMR

301

Sensory Analyses. A total of 17 panelists (nine female, eight male, 23-40 years

302

in age), who had no history of known taste disorders and who had given the informed

303

consent to participate in the present sensory tests, were trained in weekly training ACS Paragon Plus Environment

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sessions for at least two years using solutions of purified reference compounds in

305

order to become familiar with the taste language and methodologies like the so-

306

called half-tongue test.11,12,17 Sensory analyses were performed in a sensory panel

307

room at 22-25°C while the panelists wore nose clips to prevent cross-model

308

interactions with olfactory cues.

309

Taste Profile Analysis. Aliquots of the lyophilized hazelnut fractions I-III were

310

dissolved in their “natural” concentration ratios (~1.5 g powdered hazelnuts per mL)

311

in 3% ethanolic water and, then, presented to the sensory panelists who were asked

312

to rate the taste qualities “bitter”, “astringent” and “sweet” on a scale from 0 (not

313

detectable) to 5 (strongly detectable).

314

Spiking Experiment. The lyophilized methanol/water extracts prepared from

315

Cimiciato-infected hazelnuts (CN) and premium nuts (PN), respectively, were

316

dissolved in 0.3 % ethanolic water and presented in three individual sessions to the

317

trained sensory panel to evaluate the bitterness intensity on a scale from 0 (not

318

detectable) to 5 (strongly detectable). In addition, the lyophilized methanol/water

319

extract of premium nuts (PN) was added with an aliquot of the lyophilized ethyl

320

acetate fraction II isolated from Cimiciato-infected hazelnuts (CN), dissolved in 0.3%

321

ethanolic water and, then, this spiked sample (PN + fraction II) was compared to the

322

solution of the lyophilized methanol/water extract of Cimiciato-infected hazelnuts

323

(CN) on a scale from 0 (not detectable) to 5 (strongly detectable). Sensorial

324

experiments were evaluated using a touch screen and data acquisition was

325

performed using the FIZZ software (version 2.46 A; Biosystemes, Dijon, France).

326

Taste Dilution Analysis (TDA). Aliquots of MPLC fractions II-1 to II-12 and the

327

HPLC subfraction II-7-9, II-4-4, II-6-5, and II-6-6, respectively, were dissolved in

328

“natural” ratios in bottled water (35 mL), HPLC subfraction II-10-1 was dissolved in

329

1% ethanolic water (35 mL), aqueous serial 1+1 dilutions of each of these fractions ACS Paragon Plus Environment

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were prepared and presented to the sensory panel in order of ascending

331

concentration to evaluate the taste qualities “bitter, “astringent” and “sweet” of each

332

dilution. The dilution at which a taste difference between the diluted extract and the

333

blank (control) could just be detected, was defined as taste dilution (TD) factor.37 The

334

TD-factors for each HPLC-fraction, evaluated in two independent sessions each were

335

averaged. The TD-factors between individuals and separate sessions did not differ

336

by more than plus/minus one dilution step.

337

Human Taste Recognition Thresholds in Water. The threshold concentration, at

338

which the taste quality of the compound was just detectable, was determined in

339

bottled water using a three-alternative forced choice test (3-AFC) with ascending

340

concentrations of the purified test compounds (4-6). The threshold values of the

341

sensory group were approximated by averaging the threshold values of the panelists

342

in two separate sessions. The values between individuals and between the

343

independent sessions differed by not more than plus or minus one dilution step, that

344

means, i.e. the bitter threshold value of 68 µmol/L for 6 represents a range from 34 to

345

136 µmol/L.

346

Recognition Thresholds in Hazelnut Mark. To determine the bitter recognition

347

threshold of the key bitter compound 1 in hazelnut matrix, asadanin was dissolved in

348

3% ethanolic water, serial 1+1 dilutions in 3% ethanolic water were prepared and

349

aliquots (1 mL) of each dilution were added to a portion (10 g) of fresh hazelnut mark.

350

As blank (control), 10 g of the hazelnut mark was spiked with an aliquot (1 mL) of 3%

351

ethanolic water. After homogenization, the samples were presented to the trained

352

sensory panel using a two-alternative forced choice test (2-AFC) in order of

353

ascending concentration of 1 as detailed above. The threshold was determined as

354

geometrical mean of all panelists. The values between individuals and between the

355

three independent sessions differed by not more than plus or minus one dilution step. ACS Paragon Plus Environment

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Half-Tongue Test: To determine human taste recognition threshold for

357

astringency, the “half-tongue” test was applied using an ascending concentration

358

series following the procedure reported earlier.28,29 The geometric mean of the first

359

correctly answered level with all higher levels also correct andthe concentration just

360

below that concentration was calculated and taken as the individual recognition

361

threshold.11

362

Preparation of Sterile Needle Punctured (SN) and Microbially Infected Nuts

363

(MN). Sterile-needle punctured nuts (SN) were obtained by puncturing fresh hazelnut

364

kernels using a sterile 0.6 x 30 mm hypodermic needle (Sterican® Size 14, B. Braun,

365

Melsungen, Germany). Three punctures into the cotyledons down to a depth of

366

approximately 3 mm were applied to simulate mechanical damage during bug

367

feeding. Each hazelnut was treated identically, following the same pattern of

368

punctures. The punctured kernels were kept at 16 °C for 8 days and, then, stored at -

369

18 °C until analysis.

370

For the preparation of microbially infected, needle-punctured hazelnuts (MN),

371

intact hazelnuts, which were previously stored at room temperature for ten months

372

and did not show any indication of a damage, were selected, surface sterilized using

373

ethanol (70%) and, then, inoculated with individual strains of Gram-positive and

374

Gram-negative bacteria, molds and yeasts, respectively. The microorganisms used

375

for these experiments were isolated from fresh Turkish hazelnut samples as follows:

376

the microorganisms were isolated from the cupule of the collected hazelnut material,

377

which was cut into small pieces using a sterile scalpel. An aliquot (1.0 g) of this

378

material was transferred into a test tube containing a sterile, aqueous 0.9% NaCl

379

solution (9 mL), vortexed (2 min), and then serially diluted with sterile 0.9% NaCl

380

solution (up to 10-6). Aliquots (100 µL) of each dilution were spread plated on Petri

381

dishes containing plate count agar (pH 7.0) for the isolation of bacteria or malt extract ACS Paragon Plus Environment

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agar (pH 5.6) for the isolation of yeasts and molds (ROTH, Karlsruhe, Germany). The

383

Petri dishes were incubated at 30 °C for 2 days, the colonies grown were isolated by

384

single colony picking and streak plating on the corresponding culture media to obtain

385

pure cultures which were grown in agar slants and, then, stored at 4 °C until further

386

use. The following isolated microorganisms were identified by rRNA sequencing

387

(Eurofins Genomics, Ebersberg, Germany): Gram-positive bacteria (Bacillus

388

aryabhattai, Bacillus megaterium, Bacillus sp., Curtobacterium sp., Herbiconiux sp.,

389

Microbacterium hatanonis, Microbacterium phyllosphaerae, Microbacterium sp.,

390

Micrococcus sp., Paenibacillus amylolyticus, Pseudonocardia sp.), Gram-negative

391

bacteria (Acetobacter sp., Achromobacter sp, Epilithonimonas lactis, Erwinia sp.,

392

Luteibacter sp., Novosphingobium resinovorum, Novosphingobium sp., Pantoea

393

agglomerans,

394

Pseudomonas

395

Pseudoxanthomonas sp., Rahnella aquatilis, Rahnella sp., Rhizobium daejeonense,

396

Rhizobium sp., Stenotrophomonas rhizophila, Stenotrophomonas sp., Variovorax

397

ginsengisoli), molds (Alternaria sp., Aspergillus versicolor, Curvularia spicifera,

398

Fusarium equiseti, Fusarium sp., Penicillium sp., Phaeoacremonium mortoniae,

399

Plectosphaerella cucumerina, and Sarocladium strictum / Acremonium sp.), and a

400

yeast (Rhodotorula rubra). To prepare microbially infected, needle-punctured

401

hazelnuts (MN), first, microorganism suspensions used for inoculation were prepared

402

freshly before use by growing the microorganism of choice in the corresponding

403

liquid culture media for 2 – 4 days adjusted to the individual speed of growth at 28°C

404

on an orbital shaker (100 rpm, amplitude 50 mm), followed by dilution with aqueous,

405

sterile 0.9% NaCl solution to adjust a cell count of 1x106 cells/mL. For inoculation, a

406

sterile hypodermic needle was then dipped into a freshly prepared cell suspension of

407

the microorganism of choice before a new puncture was set, to ensure the

Pantoea graminis,

rodasii,

Pantoea

Pseudomonas

sp.,

Pseudomonas

kuykendallii,


fluorescens,

Pseudomonas

sp.,

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408

application of approximately equal amounts of microorganisms. Three punctures into

409

the cotyledons down to a depth of 3 mm were applied to three hazelnut kernels per

410

microorganism. Controls were treated with sterile culture media only. The inoculated

411

hazelnut kernels were incubated for 3 weeks at 22 °C and, then, stored at -18 °C

412

prior to analysis.

413

Preparation of Germinated Hazelnuts (GN). Batches of 25 intact hazelnut

414

kernels were stored in closed 3 L plastic bags, filled with 2 L moistened vermiculite,

415

at 4 °C in order to break seed dormancy. After 120 days, the sample bags were

416

opened and warmed to room temperature within a period of seven days and then

417

placed into a greenhouse. During a 14-day germination period in the greenhouse at

418

about 20 °C without direct sunlight, the vermiculite was kept moist to compensate for

419

humidity loss by evaporation. At the end of the germination period, a hazelnut was

420

considered germinating where an emergence of the radicle was observed.

421

LC-MS/MS Quantitation of Taste-Active Phytochemicals in Hazelnuts.

422

Development of an Extraction Procedure. To enable a complete and fast extraction,

423

the powdered hazelnuts (1 g), obtained by grinding deep-frozen kernels using a GM

424

300 type mill (Retsch, Haan, Germany) at 4000 rpm for 40 s, were extracted five

425

times with portions (10 mL) of either ethyl acetate, methanol, acetonitrile/water

426

(20/80; v/v), methanol/water (70/30; v/v), or methanol/2-propanol (70/30; v/v) at room

427

temperature. After extraction, the individual supernatants were measured using LC-

428

MS/MS.

429

Sample Work-Up. One hazelnut kernel (ca. 1 g) or an aliquot (1 g) of powdered

430

hazelnuts, obtained by grinding kernels using a GM 200 type mill (1000 rpm, 10 s),

431

was placed in a 15 mL bead beater tube (CK28_15 mL, Bertin Technologies,

432

Montigny-le-Bretonneux, France), filled with ceramic balls (2.8 mm i.d.), an aliquot

433

(250 µL) of a solution of the internal standard L-tryptophan-d5 in acetonitrile/water ACS Paragon Plus Environment

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(10/90; v/v; 2 mg/mL) and an aliquot (60 µL) of a solution of the internal standard

435

myricanol in acetonitrile/water (70/30, v/v; 1 mg/mL) were added and, then, the tube

436

was made up with methanol/water (70/30, v/v) to a total volume of 7 mL. After

437

extractive grinding (3 x 30 s with 20 s breaks; 7800 rpm) using the bead beater

438

(Precellys Homogenizer, Bertin Technologies), and centrifugation of the suspension

439

(3 min, 4000 rpm) using an Eppendorf Centrifuge 5702 (Eppendorf, Hamburg,

440

Germany), the supernatant was membrane filtered and injected into the LC-MS/MS-

441

system.

442

Calibration Curve and Linear Range. The internal standard solution of L-

443

tryptophan-d5 (2 mg/mL) in acetonitrile/water (10/90, v/v) was mixed with the analytes

444

2 and 3 in molar ratios from 0.01 to 180 keeping constant levels of L-tryptophan-

445

indole-d5 (0.049 mg/mL), followed by LC-MS analysis. A solution of myricanol

446

(1 mg/mL) in acetonitrile/water (70/30, v/v) was mixed with the analytes 1, 4 and 6 in

447

molar ratios from 0.1 to 8000 keeping constant levels of myricanol (0.0089 mg/mL).

448

After HPLC-MS/MS analysis in the MRM mode, calibration curves were prepared by

449

plotting the peak area ratios of analyte to its internal standard against concentration

450

ratios of each analyte to its internal standard using linear regression and the program

451

Multiquant (Version 3.0.2, Sciex, Darmstadt, Germany). The responses were linear

452

for chosen molar ratios and the contents of the indole acetic acid glycosides (2, 3)

453

and the diarylheptanoids (1, 4, 6) in samples were calculated using the respective

454

calibration functions, e.g. y = 7.4256x + 0.3162, R² = 0.9803 for 1; y =

455

17.099x + 1.139, R² = 0.9982 for 2.

456

Recovery. The recovery of the HPLC-MS/MS method was determined using

457

standard addition. Three defined but different concentrations of each analyte (1-4, 6)

458

as well as the internal standards were spiked to the powdered premium hazelnut

459

(PN) and worked up as detailed above. As reference sample (control), the premium ACS Paragon Plus Environment

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460

hazelnut matrix was extracted with spiked internal standards but without addition of

461

analytes. After sample work-up, the analytes were quantitated by means of LC-

462

MS/MS.

463

Interday and Intraday Precision. Two aliquots of the same hazelnut extract were

464

analyzed for 1-4 and 6 on consecutive days. The interday precision of the method

465

was determined by replicate analysis and expressed by the relative standard

466

deviation given in parenthesis: 1 (1.9%), 2 (11.6%), 3 (8.7%), 4 (11.6%), and 6

467

(25.7%). For the intraday precision, five aliquots of the same hazelnut extract were

468

analyzed on the same day. The precision of the developed method was determined

469

in replicate analysis and expressed by the relative standard deviation given in

470

parenthesis: 1 (9.6%), 2 (8.7%), 3 (4.6%), 4 (26.0%), and 6 (6.9%).

471

High Performance Liquid Chromatography (HPLC). The HPLC apparatus

472

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

473

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

474

degasser, a MD-2010 Plus type diode array detector, and a Rh 7725i type Rheodyne

475

injection valve (Rheodyne, Bensheim, Germany). Analytical separations were

476

performed on an analytical 250 x 4.6 mm i.d., 5 µm, Nucleodur Pyramid C18 column

477

(Macherey-Nagel, Düren, Germany) operated with a flow rate of 1 mL/min. Data

478

acquisition was done by means of Chrompass Chromatography Data System,

479

Version 1.9 (Jasco). Preparative separation of the fractions was performed on a

480

preparative 250 x 21 mm i.d., 5 µm, Nucleodur Pyramid C18 column (Macherey-

481

Nagel) operated with a flow rate of 20.0 mL/min. Data acquisition was done by

482

means of Chrompass Chromatography Data System, Version 1.9 (Jasco, Gross-

483

Umstadt, Germany).

484

Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LC-

485

MS/MS). A QTRAP 6500 mass spectrometer (Sciex, Darmstadt, Germany) was used ACS Paragon Plus Environment

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486

to acquire electrospray ionization (ESI) mass spectra and product ion spectra. The

487

MS/MS system was operated in the multiple reaction monitoring (MRM) mode

488

detecting negative ions at an ion spray voltage at -4500 V in the negative mode (ESI-

489

) and the following ion source parameters: curtain gas (35 psi), temperature (450°C),

490

gas 1 (55 psi), gas 2 (65 psi), collision activated dissociation (-2 V) and entrance

491

potential (-10 V). For analysis of compounds 1-4 and 6, the MS/MS parameters were

492

tuned to achieve fragmentation of the [M-H]- molecular ions into specific product ions

493

(Supporting Information). For tuning, acetonitrile/water solutions of each analyte and

494

internal standard were introduced by means of flow injection using a syringe pump.

495

The samples were separated by means of a Nexera X2 UHPLC (Shimadzu Europa

496

GmbH, Duisburg, Germany) consisting of two LC pump systems 30AD, a DGU-20A5

497

degasser, a SIL-30AC autosampler, a CTO-30A column oven and a CBM-20A

498

controller, and equipped with a 100 x 2.1 mm, 100 Å, 1.7 µm, Kinetex Phenyl-Hexyl

499

column (Phenomenex, Aschaffenburg, Germany). Operated with a flow rate of

500

0.4 mL/min using 1% formic acid in water (v/v) as solvent A and 1% formic acid in

501

acetonitrile (v/v) as solvent B, chromatography was performed with the following

502

gradient: 5% B held for 1 min, increased in 2 min to 30% B, in 9 min to 70% B,

503

increased in 1 min to 100% B, held 0.5 min isocratically at 100%, decreased in 1 min

504

to 5% B, held 5.5 min at 5% B. Data acquisition and instrumental control were

505

performed with Analyst 1.6.2 software (Sciex, Darmstadt, Germany).

506

UPLC/Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). An aliquot (1-

507

5 µL) of the analytes, dissolved in methanol/water (70/30, v/v; 10 mg/mL) or in

508

acetonitrile/water (70/30, v/v; 1 mg/mL), respectively, was injected into an Acquity

509

UPLC core system (Waters, Manchester, UK) connected to a SYNAPT G2 HDMS

510

spectrometer (Waters) operating in the positive or negative electrospray (ESI) modus

511

with the following parameters: capillary voltage (+2.0 kV), sampling cone (20 V), ACS Paragon Plus Environment

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source temperature (120 °C), desolvation temperature (450 °C), cone gas (5 L/h),

513

and desolvation gas (850 L/h). Chromatographic separations were performed on a

514

2.1 x 150 mm, 1.7 µm, BEH C18 column (Waters) operated at 45 °C with a solvent

515

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

516

formic acid in acetonitrile (solvent B): 0 min, 5% B, in 4 min to 100% B. The

517

instrument was calibrated over a mass range from m/z 100 to 1200 using a solution

518

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

519

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

520

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

521

the MassLynx software (version 4.1; Waters).

522

Nuclear

Magnetic

Resonance

Spectroscopy

(NMR).

1D/2D-NMR

523

experiments were performed on a Bruker 400 MHz with a Broadband Observe

524

BBFOplus probe (BB, 1H) and a 500 MHz Avance III spectrometer (Bruker,

525

Rheinstetten, Germany) equipped with a cryo-TCI probe (300 K). DMSO-d6 and

526

MeOD-d4 (600 µL) were used as solvents and chemical shifts are reported in parts

527

per million relative to the DMSO-d6 solvent signals:

528

3.33 ppm;

13

C-NMR: 39.52 ppm or the MeOD-d4 solvent signals (1H-NMR: 3.31 and

529

4.87 ppm;

13

C-NMR: 49.00 ppm). Data processing was performed by using Topspin

530

NMR software (version 3.2; Bruker, Rheinstetten, Germany) and MestReNova 10.0

531

(Mestrelab Research, Santiago de Compostela, Spain). For quantitative NMR

532

spectroscopy (qNMR), the spectrometer was calibrated by using the ERETIC 2 tool

533

using the PULCON methodology as reported earlier.24 The isolated signal at

534

6.27 ppm (d, J=1.65, 1H) was used for quantitation of 1, the signals at 7.44 and

535

7.30 ppm for quantitation of 2 (dd, J=13,5, 7,56 Hz, 1H and dd, J=13,6, 7,52 Hz, 1H),

536

the signal at 7.3 ppm for 3 (dd, J=8,65, 7,54 Hz ,1H), the signal at 4.74 ppm (dd,

537

J=11,43, 3,78 Hz, 1H) for 4, the signal at 7.15 ppm for 5 (t, J=7,74 Hz, 2H), and the ACS Paragon Plus Environment

1

H-NMR: 2.50 ppm and

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538

signal at 2.78 ppm for 6 (m, 2H), using a defined sample of L-tyrosine as the external

539

standard and its specific resonance signal at 7.10 ppm (m, 2H).38

540 541 542 543 544

RESULTS AND DISCUSSION

545 546

As the diarylheptanoid asadanin (1) has recently been reported as a major

547

contributor to the bitter off-taste of hazelnut kernels and was found in even higher

548

concentrations after Cimiciato-infection,17 the question arose as to which role

549

asadanin plays in the intense bitter taste of Cimiciato-infected hazelnuts and as to

550

whether additional phytometabolites exhibit any major taste activity.

551

Therefore, Cimiciato-infected hazelnuts (CN) were ground, extracted with

552

methanol/water (70/30, v/v) and, after removing the methanol in vacuum, the

553

aqueous extract was freeze-dried to deliver the total hazelnut extractables (fraction

554

CN), which were further extracted with n-pentane to provide the hydrophobic fraction

555

I, followed by ethyl acetate to deliver the solvent fraction II and the remaining

556

aqueous fraction III, respectively. To locate the key bitter compounds, the fractions I

557

to III were separated from solvent in vacuum, the residues taken up in 3%

558

hydroethanolic solution, each in its “natural” concentration ratio, and presented to a

559

trained sensory panel who was asked to rate the intensity of the descriptors “bitter”,

560

“astringent”, and “sweet” on a scale from 0 (not detectable) to 5 (intensely

561

detectable). The ethyl acetate extractables (fraction II) showed by far the highest

562

bitter impact with a score of 4.5, followed by fractions I and III evaluated with an

563

intensity of 2.0 and 0.9, respectively (Table 1). ACS Paragon Plus Environment

22

Page 23 of 56


564

To verify the primary taste impact of the bitter compounds present in fraction

565

II, a solution of the lyophilized methanol/water extract prepared from premium

566

hazelnuts (PN) and exhibiting only a weak bitter taste (1.0), was spiked with an

567

aliquot of fraction II isolated from the Cimiciato-infected nuts (CN) and was compared

568

to the solution of the lyophilized methanol/water Cimiciato hazelnut extract (CN) on a

569

scale from 0 (not detectable) to 5 (strongly detectable). The sensory panel rated the

570

binary mixture of the premium hazelnut extract (PN) and Cimiciato-infected hazelnut

571

fraction II with a bitter score of 3.8 which was even somewhat higher than the

572

solution of the Cimiciato hazelnut extract (CN) evaluated with a bitter score of 3.4

573

(Table 2). In consequence, the ethyl acetate extractables (fraction II) was concluded

574

to contain all major contributors to the bitter off-taste of Cimiciato-infected hazelnuts

575

and, therefore, the following fractionation was focused on the isolation and

576

identification of taste molecules in fraction II.

577

Activity-Guided Identification of Taste Compounds in Cimiciato-infected

578

Hazelnut Fraction II. The bitter tasting fraction II was separated by means of MPLC-

579

ELSD using RP-18 material as the stationary phase (Figure 2). The effluent was

580

collected in twelve fractions (II-1 to II-12), which were separated from solvent,

581

lyophilized and, then, taken up in the same amount of water for taste dilution analysis

582

(TDA). Among the 12 fractions, bitter taste was detectable in fractions II-5 to II-10

583

with fraction II-8 evaluated with the highest TD-factor of 64 (Figure 2). Comparison of

584

chromatographic and spectroscopic data (LC-MS), followed by co-chromatography

585

with the corresponding reference substance revealed fraction II-8 to contain asadanin

586

(1), recently identified as a bitter key compound in bitter hazelnuts.17

587

However, also fraction II-7 exhibited a pronounced astringency and bitterness

588

with a TD-factor of 32 (Figure 2). While the bitter taste was primarily induced by

589

some amounts of asadanin (1), UHPLC-TOF-MS screening of fraction II-7 in negative ACS Paragon Plus Environment

23


Page 24 of 56

590

mode (ESI-) and degustation experiments exhibited an astringent compound with m/z

591

541.1 [M-H]- as the pseudomolecular ion, which did not match to any phytometabolite

592

previously reported in hazelnut kernels. Therefore, fraction II-7 was separated by

593

means of preparative RP18-HPLC to afford a total of 19 subfractions, which were

594

collected individually (fractions II-7-1 to II-7-19) and analyzed again by UHPLC-TOF-

595

MS to locate the target compound (2) in fraction II-7-9. LC-MS (ESI-) analysis of the

596

purified compound 2 revealed m/z 541.1 as the pseudomolecular ion ([M-H]-), thus

597

suggesting a molecular mass of 542.1 Da and fitting well to an empirical formula of

598

C26H26 N2O11.

599

1

H and

13

C NMR data confirmed the existence of a total of 26 carbon atoms

600

and 26 protons, which could be assigned to the 2-(3-hydroxy-2-oxoindolin-3-yl) acetic

601

acid 3-O-6´-galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate (2, Figure 1). Signals

602

with chemical shifts expected for a monosaccharide could be detected in the 1H NMR

603

spectrum and indicated a glycosidic group. The signal of the protons could be related

604

to their connected carbons via 1JC,H couplings by means of heteronuclear single-

605

quantum correlation spectroscopy (HSQC). Heteronuclear multiple-bond correlation

606

spectroscopy (HMBC), optimized for 2JC,H and 3JC,H couplings, revealed finally the

607

precise structure. Due to the identical structure of the two aglycons, only differently

608

linked with the glycoside, similar chemical shifts could be detected.

609

The protons resonating between 7.47 and 6.81 ppm were correlated to the

610

carbons of the two phenyl ring systems H-C(4/4´´), C(6/6´´), C(5/5´´), and C(7/7´´).

611

The keto groups of the indole rings could be detected at 181.3 (C(2´´)) and

612

179.2 ppm (C(2)). Additionally, a 3JC,H coupling between C(3) at 80.9 ppm as well as

613

C(7a) at 144.2 ppm with H-C(4) at 7.44 ppm and a 3JC,H coupling between C(3´´) at

614

43.4 ppm and C(7a´´) at 143.6 ppm with H-C(4´´) at 7.3 ppm were observed. The

615

3

JC,H coupling between C(3) and the protons H-C(8α) at 3.15 ppm and H-C(8β) at ACS Paragon Plus Environment

24

Page 25 of 56


616

3.00 ppm as well as the 3JC,H coupling observed between C(3´´) and the protons H-

617

C(8´´α) at 3.10 ppm and H-C(8´´β) at 2.85 ppm verified the proposed structure of the

618

aglycon. The optimized

619

(homonuclear single-quantum correlation spectroscopy) between the aromatic

620

protons H-C(4) and H-C(5) at 6.94 ppm, H-C(5) and H-C(6) at 7.21 ppm, H-C(4´´)

621

and H-C(5´´) at 7,01 ppm, as well as H-C(6/6´´) at 7,21 ppm with H-C(7) and H-

622

C(7´´), resonating between 6.90 and 6.84 ppm, confirmed the two aromatic ring

623

systems (Supporting Information).

3

JH,H couplings observed in the COSY spectrum

624

One aglycon was linked via the hydroxy group of C(1´), resonating at

625

100.1 ppm, over a β-glycosidic bond as confirmed by the coupling (J=7.7 Hz)

626

between C(3) with H-C(1´), observed at 4.31 ppm (Figure 3). The second aglycon

627

was linked to a sugar moiety via its carboxy group C(9´´), resonating at 172.5 ppm.

628

The

629

3.99 ppm, as well as the 2JC,H coupling between C(9´´) and H-C(8´´), resonating

630

between 3.11 and 2.87 ppm, be observed as depicted in Figure 3.

3

JC,H coupling between C(9´´) and H-C(6´), resonating between 4.3 and

631

Taking all of the spectroscopic data into consideration, the compound isolated

632

from fraction II-7-9 could be unequivocally identified as 2-(3-hydroxy-2-oxoindolin-3-

633

yl) acetic acid 3-O-6´-galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate (2, Figure 1).

634

This phytometabolite, which to the best of our knowledge has not yet been reported

635

earlier in hazelnuts, exhibited an astringent taste above a recognition threshold

636

concentration of 400 µmol/L (Table 3).

637

The structure of phytometabolite 2 prompted us to study whether similar

638

compounds with only one indol-type aglycon exist in hazelnuts. UHPLC-TOF-MS

639

screening revealed a compound with m/z 368.1 as the pseudomolecular ion ([M-H]-)

640

and an empirical formula of C16H19NO9 in fraction II-4, which was further separated

641

by means of RP-HPLC to give subfractions II-4-1 to II-4-19, amongst which the target ACS Paragon Plus Environment

25


Page 26 of 56

642

compound was located in fraction II-4-4, isolated and analyzed by LC-MS/MS and

643

1D/2D-NMR to determine its chemical structure as 3-(O-β-D-glycosyl)dioxindole 3-

644

acetic acid 3 (Figure 1).

645

Assignment of the 2JC,H coupling between C(3) at 80.7 ppm and the protons H-

646

C(8α) at 3.34 ppm and H-C(8β) at 3.22 ppm, as well as the 2JC,H coupling between

647

the carbonyl of the carboxy group C(9) at 172.3 ppm and the protons H-C(8α/8β) led

648

to the identification of the side chain of the indole derivative. The keto group C(2) of

649

the indole ring could be observed at 178.76 ppm. In addition, a 3JC,H coupling

650

between C(3) and the anomeric proton H-C(1´), resonating at 4.18 ppm, could be

651

observed (Supporting Information). The 1H-NMR signal of H-C(1´) showed a duplet

652

with J=7.26 Hz, indicating a characteristic β-glycosidic bond between the aglycon and

653

the glycoside. Taking all of the spectroscopic data into account, the compound

654

isolated from fraction II-4-4 could be unequivocally identified as 3-(O-β-D-

655

glycosyl)dioxindole-3-acetic acid (3, Figure 1). Although this phytometabolite has

656

been earlier isolated from the peel of Citrus sinensis L.,36 its occurrence in hazelnut

657

kernels has not yet been reported. The isolated pure substance was evaluated by the

658

trained sensory panel to show only a slightly astringent taste above a threshold

659

concentration of 1000 µmol/L (Table 3).

660

UHPLC-TOF-MS analysis of the bitter tasting fractions II-6 and II-10 indicated

661

the presence of additional diarylheptanoids with molecular masses of 362.1, 360.1

662

and 312.1 Da. Hence, fraction II-6 was separated into nine subfractions (II-6-1 to II-6-

663

9) using preparative RP18-HPLC, followed by UHPLC-TOF-MS screening indicating

664

subfractions II-6-5 and II-6-6 to contain diarylheptanoids with empirical formula of

665

C19H21O7 and C19H21O7, respectively, which were purified by re-chromatography and

666

identified

as

3,8,9,10,11,12,17-heptahydroxy-tricyclo[12.3.1.12,6]nonadeca-


26

Page 27 of 56


667

1(18),2,4,6(19),14,16-hexaene (4, Figure 1) and (E)-7,9,10,13-tetrahydroxy-1,7-

668

bis(2-hydroxyphenyl)hept-9-en-11-one 5 by means of LC-MS/MS and 1D/2D-NMR.

669

13

The

C NMR spectrum of 4 demonstrated a total of 19 carbon signals,

670

whereas in the 1H NMR spectra 15 carbon-bound proton signals were observed. By

671

means of the HSQC-spectrum, optimized for 1JC,H couplings, two methylene groups,

672

eleven methin groups and six quaternary carbon atoms could be detected. The final

673

biphenyl structure with the connected C7-alkyl chain was assigned using HMBC-

674

spectroscopy. The proton signals between 7.05 and 6.74 ppm were correlated to the

675

carbons of the two connected phenyl ring systems. The biphenyl C-C-bridge was

676

verified by

677

C(18), resonating at 6.82 and 6.74 ppm, and by

678

H-C(4) at 6.81 ppm as well as H-C(19) at 6.77 ppm. The 3JC,H coupling between

679

carbon C(13) at 36.7 ppm and H-C(15) at 7.05 ppm, plus the 3JC,H coupling between

680

C(7) and H-C(5) confirmed the proposed C7-alkyl chain connected to one phenyl ring

681

at each end. In comparison to the cyclical diarylheptanoid asadanin (1), only two

682

methylene groups (C(7), C(13)) and no carbonyl group were observed in the C7-alkyl

683

chain.

3/4

JC,H couplings between C(2) at 127.4 ppm and H-C(16) as well as H3/4

JC,H couplings between C(1) and

684

To determine the structure of the alkyl chain, 3JH,H couplings between H-C(7),

685

resonating at 2,98 ppm, and H-C(8) at 4.74 ppm, between H-C(10) at 4.18 ppm and

686

H-C(11) at 4.01 ppm and, finally, between H-C(12) at 4.26 ppm and H-C(13) at

687

3.02 ppm could be detected in the COSY spectrum (Figure 4). The centrally

688

arranged carbon atom C(10) with a chemical shift of 79.52 ppm showed 3JC,H

689

couplings to the methin protons H-C(8) and H-C(12), as well as 2JC,H coupling to H-

690

C(9) (Figure 4). In addition, a 3JC,H coupling could be observed between C(9) at

691

70.1 ppm and H-C(7). Due to the suggested empirical formula of C19H22O7, each of

692

the five

13

C-atoms was assigned to be directly connected to a hydroxy group. ACS Paragon Plus Environment

27


Page 28 of 56

693

Moreover, the chemical shifts of the phenyl carbons C(3) and C(17), both observed

694

at 152.8 ppm, exhibited directly connected hydroxy groups as well. Taking all these

695

data into account, the analyzed compound could be identified as 3,8,9,10,11,12,17-

696

heptahydroxy-tricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexaene,

697

(Figure 1). Although compound 4, also known as giffonin P, has been identified

698

earlier in hazelnut leaves and hazelnut bark.25 Its occurence in hazelnut kernels as

699

well as its bitter taste activity with a taste recognition thresholds of 174 µmol/L has

700

not yet been reported. The 1H and

701

13

4

C NMR spectra of 5, showing seven proton signals and ten

702

carbon signals, together with the molecular mass of 360.1 Da and the empirical

703

formula of C19H20O7 indicated a symmetric molecule. The protons correlated to the

704

carbons of the two phenyl ring systems could be detected at chemical shifts from

705

7.22 to 6.80 ppm, namely H-C(2-5) and H-C(16-19). The coupling patterns of the

706

phenyl ring protons indicated two ortho substituted phenols. H-C(2)/H-C(16) and H-

707

C(5)/H-C(19) showed duplets with J=7.85 Hz and J=7.34 Hz, and H-C(3)/H-C(17)

708

and H-C(4)/H-C(18) pseudo triplets with J=7.74 Hz and J=7.53 Hz. Differing from

709

diarylheptanoid 4, the HMBC spectrum of compound 5 did not show any C-C-

710

correlation between the phenyl rings, thus implying a linear diarylheptanoid structure.

711

The connectivity of both phenol moieties to the C7-alkyl chain was verified by the

712

3

713

C(19), observed at 6.91 ppm, as well as by the 2JC,H couplings between C(7)/C(13)

714

and the methylene groups H2-C(8) and H2-C(12), resonating at 2.87 ppm (H-C(8α),

715

H-C(12α)) and 2.66 ppm (H-C(8β), H-C(12β)) (Supporting Information). Moreover,

716

the carbonyl C(11), resonating at 178.2 ppm and located within the C7-alkyl chain,

717

showed 3JC,H couplings to H-C(8) and H-C(12). Also the quaternary

718

could be detected at 178.2 ppm, due to the keto-enol tautomerism at carbon atoms

JC,H couplings between C(7)/C(13), resonating both at 41.8 ppm, and H-C(5)/H-


13

C-atom C(9)

28

Page 29 of 56


719

C(9) - C(11). The location in the C7-carbon chain could be confirmed considering the

720

3/4

721

Information). Taking all of the spectroscopic data into account, linear diarylheptanoid

722

5 could be determined as (E)-7,9,10,13-tetrahydroxy-1,7-bis(2-hydroxyphenyl)hept-9-

723

en-11-one (Figure 1), which to the best of our knowledge has not been reported

724

earlier. Sensory evaluation revealed this compound to exhibit bitter taste and

725

astringency above threshold concentrations of 426 and 214 µmol/L.

JC,H coupling between C(10) and H-C(8)/H-C(12) and H-C(7)/H-C(13) (Supporting

726

UHPLC-TOF-MS (ESI-) analysis and HPLC-degustation of fraction II-10 and

727

subfractions (II-10-1, II-10-2) prepared thereof revealed a bitter tasting compound (6)

728

with m/z 311.1 as the pseudomolecular ion ([M-H]-), suggesting an empirical formula

729

of C19H21O4. The 1D-NMR spectra of 6 isolated from fraction II-10-1 indicated the

730

existence of another diarylheptanoid with a basic molecule structure similar to that of

731

asadanin (1) and giffonin P (4). Instead of a biphenyl link as found for 1 and 4,

732

compound 6 comprised two phenyl ring systems connected via an O-ether bridge as

733

indicated by the chemical downfield shifts of C(1) and C(2), resonating at 148.9 and

734

148.6 ppm, as well as by the 3/4JC,H couplings between C(1) and the proton signals at

735

6.91 ppm (H-C(18)), 6.81 ppm (H-C(16)), and 6.53 ppm (H-C(19)), as well as the

736

3/4

737

and H-C(19), respectively (Supporting Information). The connection of the hydroxy

738

group to C(15), resonating at 153.5 ppm, was confirmed by the 3JC,H coupling to the

739

protons H-C(13) and H-C(18). Carbon C(3), resonating at 146.1 ppm, however,

740

showed a 3JC,H coupling to H-C(5), observed at 6.59 ppm (Supporting Information).

741

Like the other diarylheptanoids 1, 4 and 5, the two phenyl rings of 6 were connected

742

via a C7-alkyl chain as supported by the 3JC,H couplings between C(7) (131.1 ppm)

743

and H-C(5) (6.59 ppm) as well as a 3JC,H coupling between C(13) (28.9 ppm) and H-

744

C(18) (6.91 ppm), respectively. In addition, the alkyl chain in 6 was found to be

JC,H couplings observed between carbon C(2) and the protons H-C(4), H-C(18),


29


Page 30 of 56

745

decorated with a double bond and a hydroxy group. The double bond could be

746

unequivocally identified in the COSY spectrum showing a 3JH,H coupling between H-

747

C(7) and H-C(8) with a coupling constant of J=11.35 Hz as typically found for a cis-

748

configured double bond. Additional 3JH,H couplings could be detected between H-C(8)

749

and H-C(9), H-C(9) and H-C(10), H-C(11) and H-C(12), and between H-C(12) and H-

750

C(13) resulting in the final structure of the alkyl chain (Supporting information).

751

Taking all of the spectroscopic and spectrometric data into consideration, the

752

phytometabolite 6, isolated from fraction II-10-1, could be unequivocally identified as

753

4,12,16-trihydroxy-2-oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-

754

heptaene (Figure 1). To the best of our knowledge this compound has not been

755

reported before in literature. Sensory evaluation revealed an intense bitter taste

756

above a taste threshold concentration of 68 µmol/L.

757

To investigate the contribution of the identified phytochemicals to the bitter

758

taste of Cimiciato-infected hazelnuts, the bitter-tasting diarylheptanoids 1, 4, and 6

759

and the astringent 3-(O-β-D-glucosyl)-2-oxo indole-3-acetic acids 2 and 3 were

760

quantitated in a series of well-defined hazelnut samples by means of LC-MS/MS. As

761

the linear diarylheptanoid 5 did not show an intense bitter taste (threshold

762

426 µmol/L) compared to the cyclical diarylheptanoids (thresholds 13-174 µmol/L),

763

and has therefore, if any, only marginal effects on the bitter off-taste of hazelnuts, it

764

was not included in the following quantitative analysis.

765

Method Development for Quantitation of Taste-Active Phytometabolites

766

(1-4, 6) in Hazelnuts. To enable the fast and reliable quantitation of the

767

phytometabolites identified in a large sample set, a fast extraction and sample work-

768

up procedure was developed using a bead beater for combined homogenization and

769

extraction within 3 min and, after internal standard addition and equilibration (30 min),

770

LC-MS/MS analysis was performed using L-tryptophane-d5 as the internal standard ACS Paragon Plus Environment

30

Page 31 of 56


771

for quantitation of the target analytes 2 and 3 and myricanol for the bitter compounds

772

1, 4, and 6 (Figure 5). As the evaluation of a series of solvent mixtures revealed

773

methanol/water (70/30, v/v) to show best extraction results, this solvent mixture was

774

used for all quantitative studies. MS/MS parameters were tuned for each analyte (1-

775

4, 6) and both internal standards to optimize the generation of specific product ions of

776

the pseudo molecular ion through fragmentation, by individually infusing the

777

corresponding reference compounds with a syringe pump into the mass

778

spectrometer using the ESI- mode. The most sensitive ion transition for each analyte

779

was selected for quantitation and a second mass transition was used as qualifier for

780

unequivocal identification (Figure 5).

781

To check the accuracy of the analytical method, recovery and sensitivity of the

782

quantitation method were determined. Aliquots of powdered premium hazelnuts (PN)

783

were, therefore, spiked in three different concentration levels (0.1 to 53.1 µmol/L)

784

with the target compounds (1-4, 6) prior to quantitative analysis, using two powdered

785

premium hazelnut samples without additional spiking as control samples.

786

Comparison of the amounts determined with those found in the blank hazelnut

787

samples (control) exhibited satisfying recoveries of 97% (1) 126.5% (2), 108.6% (3),

788

87.2% (4), and 119.5% (6), respectively. Repeatability of the method was

789

demonstrated by analysis of three different hazelnut samples for three times, e.g. the

790

coefficient of variation ranged between 15 (3) and 20% (2). Intra-assay precision was

791

determined by analysis of one batch of hazelnuts in five replicates and demonstrated

792

a low coefficient of variation between 4 to 25%. In addition, analysis of one batch of

793

hazelnuts on two independent days was performed to determine the inter-assay

794

precision and showed a low coefficient of variation ranging from 1.9 to 25% for the

795

analytes 1-4 and 6. After validation, this method was used to investigate the factors

796

driving the biosynthesis of the taste compounds in hazelnuts. ACS Paragon Plus Environment

31


797

Influence

of

a

Cimiciato-Infection

on

the

Page 32 of 56

Concentration

of

798

Phytometabolites 1-4 and 6 in Hazelnuts. A total of 87 samples of hand-selected

799

premium hazelnuts (PN), lacking any physical damage and bitter off-taste, and 33

800

samples of Cimiciato-infected hazelnuts (CN) were analyzed using the LC-MS/MS

801

method developed above (Figure 6). All the diarylheptanoids 1, 4, and 6 were found

802

in significantly higher amounts in the Cimiciato-infected hazelnuts compared to the

803

premium hazelnut samples (Figure 6, A-C). In particular, asadanin (1, Figure 6A)

804

was present in highly increased amounts of up to a maximum of 526 µmol/kg

805

(140 µmol/kg on average) in CN when compared to PN with an average level of

806

12 µmol/kg. In a minor number of 17 out of the 87 analyzed PN samples,

807

comparatively high diarylheptanoid levels were found, shown as dots in Figure 6;

808

these may be explained by an undetected early-stage Cimiciato-infection or by

809

another

810

biosynthesis. When compared to the diarylheptanoids, interestingly, the amounts of

811

the indole derivatives 2 and 3 were not affected by a Cimiciato-infection (Figure 6, D

812

and E) and only marginal concentration differences were found between the

813

samples.

factor

driving

metabolic

alteration

and

facilitated

diarylheptanoid

814

To gain some further insight into the cause of a stimulated diarylheptanoid

815

biosynthesis upon Cimiciato infection, quantitative model experiments were

816

performed in the following. To imitate a bug bite and to study whether or not the

817

physical damage triggers an abiotic stress reaction resulting in an increased

818

diarylheptanoid generation, first, surface-sterilized hazelnut kernels, which were

819

previously stored at room temperature for ten months and did not show any

820

indication of a damage, were punctured with a sterile hypodermic needle to simulate

821

mechanical damage during bug feeding. After keeping the sterile-needle punctured


32

Page 33 of 56


822

hazelnut kernels (SN) at 16 °C for 8 days, the analytes 1-4 and 6 were analyzed by

823

means of LC-MS/MS.

824

To imitate the potential transfer of microorganisms to the hazelnut kernel

825

during the bug attack and to study whether or not the increased diarylheptanoid

826

generation is due to a biotic stress response, the bug infection was mimicked in the

827

lab in a series of experiments. To achieve this, intact, surface-sterilized hazelnut

828

kernels were punctured with a hypodermic needle which was dipped into a freshly

829

prepared cell suspension (1x106 cells/mL) of individual microorganisms out of a

830

collection of 11 Gram-positive and 22 Gram-negative bacteria, nine molds and one

831

yeasts, which were isolated from hazelnut samples collected in Turkey, identified by

832

rRNA sequencing, and cultured accordingly. The microbially infected hazelnut

833

kernels (MN) were kept for 3 weeks at 22 °C and, then, the analytes 1-4 and 6 were

834

analyzed by means of LC-MS/MS.

835

Interestingly, the sterile-needle punctured hazelnuts (SN) and microbially

836

infected hazelnut samples (MN) did not show any significant differences in

837

diarylheptanoid contents (1, 4, 6) when compared to the reference premium

838

hazelnuts (PN), e.g. average amounts of 1 were 1 µmol/kg in SN samples and

839

5 µmol/kg in MN samples and were in the same range as found for the premium nuts

840

(PN, 12 µmol/kg) (Figure 6). Also the amount of the indole derivatives (3, 2) were

841

almost identical in samples PN, SN and MN (Figure 6). These studies demonstrate

842

that it is not the physical damage alone and also not a general microbial infection that

843

may cause a stress-induced generation of diarylheptanoids, but most likely either

844

specific Cimiciato-specific microorganisms associated with the bugs,26,29,31 or

845

possible elicitors like proteases, esterases, and lipases in the bugs’ saliva secreted

846

upon infection of the nuts.10,29–32 This needs to be further clarified by more

847

sophisticated future studies. ACS Paragon Plus Environment

33


Page 34 of 56

848

In addition to the Cimiciato-infection, also germination processes are well-

849

known to alter metabolism in the hazelnut kernel.33–35 The following quantitative

850

experiments were, therefore, performed to study whether metabolic changes during

851

an early phase of germination of the hazelnuts play a role as a candidate trigger of

852

arylheptanoid biosynthesis.

853

Influence of Germination on the Concentration of Phytometabolites 1-4

854

and 6 in Hazelnuts. Hazelnut seeds can be in a dormant state, which is induced

855

during drying and is characterized by a shut-down of metabolism and resulting in a

856

higher storage stability of the hazelnut kernels, and a germinable state, which can be

857

induced by cold charming (0-7 °C) of hazelnuts in dormancy and lead to drastic

858

changes in metabolic activity.33–35 The physiological regulation of dormancy and

859

germ induction involves different seed tissues and, in particular, the germ-axis which

860

can be affected in its viability and, in consequence, in its metabolic activity by high

861

temperatures during drying or fast and intense dehydration.39

862

To gain first insight into the effect of germination on arylheptanoid generation,

863

model germination experiments were performed with hazelnut kernels. To achieve

864

this, intact hazelnut kernels were stored first at 4 °C in order to break seed dormancy

865

and, after 120 days, then kept at room temperature for 7 days, followed by a 14-day

866

germination period in the greenhouse at 20 °C without direct sunlight. A total of 150

867

germinated hazelnut kernels (GN) were then analyzed by means of LC-MS/MS in

868

triplicates and compared to non-germinated, premium hazelnuts (PN) as reference

869

(Figure 7). The quantitative data clearly demonstrated a stimulated biosynthesis of

870

diarylheptanoids upon germination with all diarylheptanoids (1, 4, 6) detected in

871

significantly higher levels in the GN samples (Figure 7). The highest levels were

872

found for asadanin (1), the key contributor to the bitter off-taste in hazelnuts,17 e.g.

873

enormous concentrations of up to ~2000 µmol/kg were found in GN samples when ACS Paragon Plus Environment

34

Page 35 of 56


874

compared to the PN samples showing maximum amounts of 50 µmol/kg. Again, the

875

indole derivatives (3, 2) were not affected to a major extend by germination, e.g. the

876

concentrations about 2000 µmol/kg (3) and 500 µmol/kg (2) in GN were similar to

877

those found in premium hazelnut samples (Figure 7).

878

Consequently, not only the Cimiciato-infection but also the germination of the

879

hazelnut kernel has been shown for the first time to activate diarylheptanoid

880

biosynthesis, resulting in higher contents of bitter tasting phytochemicals and

881

development of the bitter off-taste.

882

Dose/Activity

Considerations

on

the

Sensory

Impact

of

the

883

Diarylheptanoids 1, 4 and 6 in Hazelnuts. To visualize the contribution of the

884

individual diarylheptanoids to the bitter-off taste in hazelnuts, distribution graphs

885

illustrating the content of phytometabolites 1, 4, and 6 in 87 premium (PN), 33

886

Cimiciato-infected (CN), and 150 germinated hazelnut samples (GN) were calculated

887

and compared to the bitter taste recognition threshold of 13, 174, and 68 µmol/L

888

found for 1, 4, and 6 in water (Table 3). In addition, a bitter breakthrough threshold

889

concentration of 37 µmol/kg was determined for asadanin (1) in fresh hazelnut mark.

890

As depicted in Figure 8, the concentrations of the diarylheptanoids in premium nuts

891

(PN) were always below the threshold level and well in agreement with the absence

892

of any perceivable bitter off-taste. In comparison, the maxima of the distribution

893

curves calculated for asadanin (1) in Cimiciato-infected (CN) and germinated nut

894

samples (GN) exceeded the bitter threshold concentration of 13 (water) and

895

37 µmol/kg (hazelnut mark) respectively. As the concentration of the diarylheptanoids

896

4 and 6 did not exceed their threshold concentrations, these data unequivocally

897

demonstrate asadanin (1) as the key contributor to the bitter off-taste in Cimiciato-

898

infected as well as germinated hazelnuts (Figure 8). Consequently, asadanin (1) can


35


Page 36 of 56

899

be considered the most promising candidate to develop an objective analytical quality

900

control of hazelnuts and products containing them.

901 902

Acknowledgment

903

This IGF Project of the FEI was supported via AiF within the program for promoting

904

the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs

905

and Energy (BMWi), based on a resolution of the German Parliament. We

906

acknowledge the technical support by Luis Fernando Izaguirre de Leon, Dr. Cornelia

907

Koob, and Hasnaa Ibrahim during hazelnut inoculation experiments. The authors

908

thank SCIEX, Darmstadt, Germany, for providing technical support.

909 910

SUPPORTING INFORMATION AVAILABLE

911

NMR spectrometric data, LC-MS parameters, and HPLC chromatograms. This

912

material is available free of charge via the Internet at http://pubs.acs.org.

913


36

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914

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kernel feeding by Halyomorpha halys (Hemiptera: Pentatomidae) on

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commercial hazelnuts, J. Econ. Entomol. 2014, 107, 1858–1865.

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hazelnut kernel quality in Turkey; Intern. Soc. Hortic. Sci. (ISHS), Leuven,

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and sterols of hazelnut oil, Europ. Food Res. Technol. 2016, 243, 651-658.

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Heß, D. Plant physiology, Ulmer, Stuttgart. 2008.


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tasting 1H,4H-quinolizinium-7-olate by application of the taste dilution analysis,

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compounds in foods, J. Agric. Food Chem. 2001, 49, 231–238.

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spectroscopy, J. Agric. Food Chem. 2014, 62, 2506–2515.

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Kermode, A. R.; Gifford, D. J.; Bewley, J. D. The role of maturation drying in

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1023

seeds, J. Exp. Bot. 1985, 36, 1928–1936.

1024 1025 1026


41


Table 1.

Page 42 of 56

Taste Profile Analysis of Fractions I-III isolated from Cimiciato-infected Hazelnut Kernels Exhibiting a Bitter Off-Taste.

Fractiona

Intensityb perceived for bitterness

astringency

sweetness

I

2.5

1.7

0.5

II

4.5

2.3

0.2

III

0.9

3.4

2.9

a

The n-pentane soluble fraction I, the ethyl acetate soluble fraction II, and the

aqueous fraction III isolated from Cimiciato-infected hazelnuts were dissolved in 3% ethanolic water in their “natural” concentrations and, then used for taste profile analysis. bA trained sensory panel was asked to rate the intensity of the given taste descriptors on a scale from 0 (not detectable) to 5 (intensely detectable).


42

Page 43 of 56


Table 2. Bitter Taste Intensity of Premium Hazelnut Extract (PN), Cimiciato-infected Hazelnut Extract (CN), and PN spiked with Fraction II Isolated from CN. Sample

a

bitterness

PN

1.0

CN

3.4

PN + fraction II

3.8

a

A trained sensory panel was asked to rate the bitterness intensity of an aqueous

solution of the lyophilized methanol/water extractables prepared from premium nut samples (PN) and Cimiciato-infected nut samples (CN) on a scale from 0 (not detectable) to 5 (intensely detectable) and, then, to compare them to an aqueous solution of the lyophilized methanol/water extractables prepared from premium nut samples (PN) spiked with fraction II isolated from CN (PN + fraction II).


43


Page 44 of 56

Table 3. Taste Recognition Thresholds of Phytometabolites 1-6 in Water. Bitter threshold

Astringent threshold

concentrationa [µmol/kg]

concentration[µmol/kg]

1

13.0

≥1000b

2

≥1000b

400

3

≥1000b

≥1000b

4

174

≥1000b

5

426

214

6

68

≥1000b

Compound no.

a

Bitter threshold concentrations in water. bNo taste activity perceived up to the

highest test concentration of 1000 µmol/kg..


44

Page 45 of 56


FIGURE LEGEND

Figure 1.

Chemical structures of phytometabolites 1-6 identified in Cimiciato-infected hazelnuts.

Figure 2.

(A) RP18-MPLC separation of fraction II isolated from Cimiciato-infected hazelnut kernels and (B) taste dilution (TD)-factors of MPLC fractions II-1 to II-12.

Figure 3.

Excerpts of the HMBC spectrum (125 MHz, MeOD-d4) of 2-(3-hydroxy-2oxoindolin-3-yl) acetic acid 3-O-6´-galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate (2) with (A) highlighted 3J-coupling of C(3), and (B) 2J- and 3Jcoupling of C(9´´).

Figure 4.

Excerpts of the (A) COSY spectrum (500 MHz, MeOD-d4) with highlighted 3

J-couplings of the protons of the alkyl chain and (B) HMBC spectrum

(125 MHz, MeOD-d4) of giffonin P (4) exhibiting 2J- and 3J-coupling of the carbonyl atom C(10).

Figure 5.

HPLC-MS/MS analysis of a hazelnut sample showing the mass transitions for the quantitation of the astringent indole-type glycosides (2, 3) and the bitter diarylheptanoids (1, 4, 6) using L-tryptophane-d5 (IS1) and myricanol (IS2) as the internal standards.


45


Figure 6

Page 46 of 56

Boxplots of the concentration of phytometabolites in premium nuts (PN), Cimiciato-infected nuts (CN), sterile-needle punctured nuts (SN), and needle-punctured and microbial infected nuts (MN): (A) asadanin (1), (B) giffonin P (4), (C) 4,12,16-trihydroxy-2-oxatricyclo[13.3.1.13,7]-nonadeca1(18),3,5,7(20),8,15,17-heptaene (6), (D) 3-(O-β-D-glycosyl)dioxindol-3acetic acid (3), and (E) 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3-O-6´galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate (2).

Figure 7.

Boxplots of the concentration of phytometabolites in premium nuts (PN) and germinated nuts (GN): (A) asadanin (1), (B) giffonin P (4), (C) 4,12,16-trihydroxy-2-oxatricyclo[13.3.1.13,7]-nonadeca1(18),3,5,7(20),8,15,17-heptaene (6), (D) 3-(O-β-D-glycosyl)dioxindol-3acetic acid (3), and (E) 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3-O-6´galactopyranosyl-2“-(2“oxoindolin-3“yl) acetate (2).

Figure 8.

Distribution graph displaying the concentrations and taste threshold concentration (TC) of (A) asadanin, 1, TC: 13 µmol/L (water, slightly dashed line), 37 µmol/kg (hazelnut mark, intensely dashed line), (B) giffonin P, 4, TC: 174 µmol/L (water, dashed line), (C) 4,12,16-trihydroxy2-oxatricyclo[13.3.1.13,7]-nonadeca-1(18),3,5,7(20),8,15,17-heptaene,

6,

TC: 68 µmol/L (water, long dashed line) in premium nuts (PN, red line), Cimiciato-infected nuts (CN, blue line), and germinated nuts (GN, green line).


46

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Figure 1 (Singldinger et al.)


47


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48

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A

B


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A

B


50

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A

B


51


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52

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