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Decoding the Nonvolatile Sensometabolome of Orange Juice (Citrus sinensis) Anneke Glabasnia, Andreas Dunkel, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06142 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Journal of Agricultural and Food Chemistry

1

Decoding the Nonvolatile Sensometabolome of

2

Orange Juice (Citrus sinensis)

3 4 5

Anneke Glabasnia+, Andreas Dunkel†, and Thomas Hofmann†,‡,*

6

+

7

Münster, Germany

8

†

9

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

Institut für Lebensmittelchemie, Universität Münster, Corrensstrasse 45, D-48149

Chair of Food Chemistry and Molecular Sensory Science, Technical University of

10

‡

11

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

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

12 13 14 15 16 17 18 19

*

To whom correspondence should be addressed

20

PHONE

+49-816171-2902

21

FAX

+49-816171-2949

22

E-MAIL

[email protected]

23 24 25

1 Environment ACS Paragon Plus


26

ABSTRACT

27

Activity-guided fractionation in combination with the taste dilution analysis, followed

28

by LC-MS/MS and NMR experiments led to the identification of ten polymethoxylated

29

flavones (PMFs), six limonoid glucosides, and two limonoid aglycones as the key

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bitterns of orange juice. Quantitative studies and calculation of dose-over-threshold

31

factors, followed by taste re-engineering demonstrated for the first time 25

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sensometabolites to be sufficient to re-construct the typical taste profile of orange

33

juices and indicated that not a single compound can be considered a suitable marker

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for juice bitterness. Intriguingly, the taste percept of orange juice seems to be created

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by a rather complex interplay of limonin, limonoid glucosides, PMFs, organic acids,

36

and sugars. For the first time, sub-threshold concentrations of PMFs were shown to

37

enhance the perceived bitterness of limonoids. Moreover, the influence of sugars on

38

the perceived bitterness of limonoids and PMFs in orange juice relevant

39

concentration ranges was quantitatively elucidated.

40 41

Key Words: polymethoxylated flavones, 5,6,7,3´,4´-pentamethoxyflavone-3-O-β-D-

42

glucopyranoside, limonin, nomilin, limonoid glucosides, bitter taste, astringency, taste

43

dilution analysis, half-tongue test

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INTRODUCTION

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Due to its alluring aroma and the desirable taste profile, orange juice is one of the

48

most popular fruit juices with a global production volume of 2.4 million metric tons per

49

year.1 Besides the aroma-active volatiles, the balanced harmony of sweetness and

50

sourness accompanied by a velvety astringency and a faint bitterness are

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determining the acceptability of orange juice by the consumer.

52

Screening of orange volatiles for odor-activity by means of gas chromatography-

53

olfactometry, quantitation of the most odor-active molecules by using stable isotope

54

dilution analyses, followed by aroma reconstitution experiments impressively

55

demonstrated that only about 25 out of more than 200 volatiles contribute to the

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typical aroma of a freshly squeezed orange juice.2-5 In contrast, the knowledge on the

57

taste-active non-volatiles creating the attractive taste percept of orange juice in the

58

brain is rather fragmentary. In particular, the knowledge on the molecules imparting

59

the typical bitter taste of orange juice is rather contradictory.

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Limonin (1; Figure 1), a highly oxygenated triterpenoid of the family limonoids,

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has been suggested already in 1841 as a constituent of oranges,6 although its

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chemical structure as well as its contribution to the bitter taste of navel orange juice

63

were reported almost one hundred years later.7,8 Sensory analysis revealed a bitter

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taste threshold of 6 ppm (in orange juice) for limonin and 2 ppm (in water) for nomilin

65

(2), another member of the limonoid family.9-11 However, nomilin (2) is suggested to

66

exhibit only a minor contribution to the bitter taste of orange juice.12 Although the

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bitterness of limonin (1) is reported to be suppressed by sucrose and citric acid,9,13

68

systematic studies addressing the question as to how such mixture suppression

69

effects do quantitatively impact the perception of bitter compounds in orange juices

70

are lacking.



71

Whole orange fruits usually do not contain the bitter-tasting limonin in supra-

72

threshold concentrations but contain its precursors, namely limonoate A-ring lactone

73

and limonin-17-O-β-D-glucopyranoside (3; Figure 1). After juice preparation from

74

early to mid season harvested oranges, limonoate A-ring lactone is reported to be

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gradually converted into the bitter tasting limonin (1) by proton-catalyzed

76

lactonization and/or upon catalysis of limonoid D-ring lactone hydrolase, thus

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imparting the so-called “delayed” bitterness.14-16 Limonoid glucosides such as

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compound 3 are known to be generated from the corresponding aglycone during the

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late stages of fruit growth and maturation and are present in orange juices in an

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aglycone/glucoside ratio of about 1:150.17-19 However, data available on the sensory

81

impact of these limonoid glucosides are rather contradictory, e.g. some researchers

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suggest limonin-17-O-β-D-glucopyranoside (3) to contribute to the bitter taste of

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orange juices,20 whereas other publications report this glucoside to be tasteless at

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all.21 Model studies on the degradation of compound 3 under acidic conditions as well

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as during storage of orange juice revealed the formation of limonin (1) besides its

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epimer C17-epilimonin.22 Quantitative data showed evidence for the use of the C17-

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epilimonin/limonin ratio as a suitable marker for the analytical determination of the

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thermal input applied during orange juice manufacturing.22

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Another family of phytochemicals hypothesized in literature as candidate taste

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molecules of orange products is the group of polymethoxylated flavones with the

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general structure displayed in Figure 2. As these polyphenols are mainly located in

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the peel and, in particular, in the flavedo of the fruit,23 their concentration in orange

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juice was reported to be dependent on the extraction method used and pressure

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applied during juicing.24 The highest levels of polymethoxylated flavones were found

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in young developing fruits,25 although a relation of some of these polyphenols to the

96

maturation phase of the fruit was suggested.23 Sensory analysis of a chemically


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undefined, crude mixture of polymethoxylated flavones, isolated from orange juice

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concentrate, revealed a taste threshold of 15-46 mg/kg without giving any information

99

about the perceived taste quality.26 Overall, these flavones were concluded not to

100

contribute to the flavor of orange juice.

101

Driven by the need to discover the key players imparting the typical taste of

102

foods, the research area “sensomics” has made tremendous efforts in recent years in

103

order to analytically decode the so-called sensometabolome, defined as the

104

composition of the group of sensory active key molecules “synthesizing” the sensory

105

percept of a given food in the brain.27,28 Aimed at identifying the essential molecules

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coding the typical taste signature of orange juice, the objectives of the present

107

investigation were, therefore, to systematically identify and quantify the key

108

sensometabolites in orange juice, to functionally validate their sensory contribution by

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means of taste re-engineering experiments, and to gain some first insight into their

110

natural variation in commercial orange juices.

111 112 113 114

MATERIALS AND METHODS

115 116

Chemicals. The following compounds were obtained commercially: limonin,

117

nomilin, hesperidine (Sigma, Steinheim, Germany), scutellarein tetramethylether,

118

sinensetin (Extrasynthèse, Lyon, France); formic acid and organic solvents were of

119

HPLC

120

Taufkirchen, Germany); de-ionized water used for chromatography was purified by

121

means of a Milli-Q Gradient A10 system (Millipore, Billerica, USA). For sensory

122

analyses, bottled water (Evian, low mineralization 500 mg/L) was adjusted to pH 4.5

grade

(Merck,

Darmstadt,

Germany);

deuterated


solvents

(Aldrich,


123

with 0.1% aqueous formic acid. Juice prepared from Valencia oranges harvested on

124

30th November 2003 (sample OJ1), 13th March 2006 (sample OJ2), 10th January

125

2004 (sample OJ3), 24th February 2004 (sample OJ4), 30th March 2006 (sample

126

OJ5), respectively, were provided by the industry (USA). Orange seeds and orange

127

molasses, a concentrated thick viscous liquid and intensely bitter tasting by-product

128

of citrus juice extraction, were provided by the industry (USA). Commercial orange

129

juices (OJ6 - OJ15) were purchased at a local store in Germany. provided by the

130

industry (USA)

131

Sequential Solvent Extraction of Orange Juice (OJ1) and Orange

132

Molasses. Aliquots (100 g) of orange juice OJ1 and molasses, respectively, were

133

centrifuged for 10 min at 11269 × g (Varifuge 20 RS, Heraeus), the supernatant was

134

diluted with water (pH 4.5) to a volume of 250 mL, and, then, sequentially extracted

135

with pentane, followed by dichloromethane, and ethyl acetate (3 x 300 mL each). The

136

corresponding organic layers were combined to give the pentane solubles (fraction I),

137

the dichloromethane solubles (fraction II), and the ethyl acetate solubles (fraction III)

138

after solvent separation in vacuum, suspension of the residues in water (10 mL), and

139

freeze-drying. Freeze-drying of the remaining aqueous layer gave the water soluble

140

fraction IV. After determination of the yields (Table 1), fractions I-IV isolated from

141

orange juice OJ1 and orange molasses, respectively, were kept at -18° C until used

142

for sensory and chemical analysis.

143

HPLC Separation of Fraction II Isolated from Orange Molasses. An aliquot

144

(500 mg) of fraction II isolated from orange molasses was dissolved in a mixture

145

(50/50, v/v; 10 mL) of methanol and aqueous formic acid (0.1% in water) using an

146

ultrasonic bath. After membrane filtration (0.45 µm, Satorius Hannover, Germany),

147

aliquots (300 µL) were separated by semi-preparative HPLC on a 250 x 10 mm i.d., 5

148

µm, Microsorb–MV RP-18 column (Varian, Germany). Monitoring the effluent at 272


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149

nm, 19 fractions, namely II-1 to II-19 (Figure 3), were individually collected into ice-

150

cooled glass flasks. The corresponding fractions obtained from 15 HPLC runs were

151

combined, and after the solvent was removed in vacuum and freeze dried twice,

152

were directly used for taste dilution analysis (TDA).

153

Isolation of Taste Compounds 1, 2, and 4-13 from Fractions II-8 to II-19.

154

An aliquot (1.0 g) of fraction II isolated from orange molasses was dissolved in a

155

mixture (50/50; v/v; 10 mL) of methanol and aqueous formic acid (0.1 % in water; pH

156

2.5), membrane filtered, and aliquots (300 µL) were separated by HPLC on a 250 x

157

10 mm i.d., 5 µm, Microsorb–MV RP-18 column (Varian, Germany). Monitoring the

158

effluent at 272 nm, chromatography was performed with a mixture (20/80; v/v) of

159

acetonitrile and aqueous formic acid (0.1% in water), increasing the acetonitrile

160

content to 30% within 15 min, then to 40% within 40 min, and, finally, to 100% within

161

additional 15 min, thereafter elution with acetonitrile for 5 min at a flow rate of

162

3.5 mL/min. A total of 12 peaks evaluated with high TD-factors were collected and,

163

after separating the solvent in vacuum, the residues were freeze-dried twice to give

164

the taste compounds limonin (1), nomilin (2), 5,6,7,3´,4´-pentamethoxyflavone

165

(sinensetin, 4), 5,6,7,4´-tetramethoxyflavone (scutellarein tetramethylether, 5),

166

5,6,7,8,4´-pentamethoxyflavone (tangeretin, 6), 5,7,8,3´,4´-pentamethoxyflavone (7),

167

3,5,7,8,3´,4´-hexamethoxyflavone

168


169

3,5,6,7,8,3´,4´-heptamethoxyflavone

170

pentamethoxyflavone (13) in a purity of >99% (HPLC/DAD). LC-MS/MS and 1D/2D-

171

NMR spectroscopic data of 1, 2, and 4-13, respectively, were identical to those

172

reported earlier29-33 and are given as supplementary information.

(8),


(9),

5,7,8,4´-tetramethoxyflavone

(11),

(10), (12),

and

4´-hydroxy-5,6,7,8,3´-

173

Isolation of Taste Compounds 14 and 15 in Fractions II-3 and II-4. An

174

aliquot (1.2 g) of fraction II isolated from molasses was taken up in a mixture (40/60,



175

v/v; 15 mL) of methanol and aqueous formic acid (0.1% in water) and was applied

176

onto the top of a water-cooled 300 x 35 mm i.d. glass column, which was filled with a

177

slurry of RP-18 material (25-40 µm) conditioned with methanol/0.1% aqueous formic

178

acid (40/60, v/v). Chromatography was performed with a flow rate of 2 mL/min using

179

mixtures of methanol and 0.1% aqueous formic acid to give fractions II-A (40/60, v/v;

180

100 mL), II-B (50/50, v/v; 100 mL), II-C (60/40, v/v; 100 mL), II-D (70/30, v/v;

181

100 mL), II-E (80/20, v/v; 100 mL), followed by methanol (100 mL) and acetonitrile

182

(100 mL) to afford fractions II-F and II-G, respectively. After separating the individual

183

fractions from solvent in vacuum, the residues were analysed by analytical RP-

184

HPLC. Fractions II-A to II-D, containing the taste compounds located in HPLC/TDA

185

fractions II-3 and II-4, were combined, taken up in water (20 mL) and separated by

186

gel adsorption chromatography (GAC) on a 380 x 40 mm i.d. column (Amersham

187

Pharmacia Biotech, Sweden) filled with a slurry of Sephadex LH-20 (Amersham

188

Pharmacia Biotech) in 0.1% aqueous formic acid. Chromatography was performed at

189

a flow rate of 2 mL/min starting with aqueous formic acid (0.1% in water, 250 mL),

190

followed by mixtures of methanol and 0.1% aqueous formic acid: 20/80 (v/v, 250 mL),

191

40/60 (v/v, 250 mL), 60/40 (v/v, 250 mL), and 80/20 (v/v, 250 mL). Monitoring the

192

effluent at 272 nm, a total of 12 GAC fractions were collected (30 min each),

193

separated from solvent in vacuum, and freeze dried twice. GAC fractions no. 3 and 4,

194

containing the taste compounds located in HPLC/TDA fractions II-3 and II-4, were

195

dissolved in a mixture (50/50; v/v) of methanol and aqueous formic acid (0.1% in

196

water), membrane filtered, and aliquots (250 µL) were separated by means of semi-

197

preparative HPLC on a 250 x 10 mm i.d., 5 µm, RP-18 column (Varian, Germany).

198

Monitoring the effluent at 272 nm, chromatography was performed at a flow rate of

199

3.5 mL/min with a mixture (10/90, v/v) of acetonitrile and aqueous formic acid (0.1%

200

in water), increasing the acetonitrile content to 20% within 10 min, holding for 20 min,


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201

then increasing to 30% within 20 min, and, finally, to 100% within 2 min. The two

202

main peaks were collected, the solvents separated in vacuum, and the remaining

203

residue

204

pentamethoxyflavone-3-O-ß-D-glucopyranoside

205

hexamethoxyflavone-3-O-ß-D-glucopyranoside

206

(HPLC/ELSD, HPLC/MS, 1H NMR). The spectroscopic data of 15 were well in line

207

with those reported earlier34 and are given as supplementary information.

208

5,6,7,3´,4´-Pentamethoxyflavone-3-O-β-D-glucopyranoside,

of

each

fraction

was

freeze-dried

twice

to

(14) (15)

in

give

and a

5,6,7,3´,4´5,6,7,8,3´,4´-

purity

14,

of

>

99%

Figure

2:

209

LC/MS (ESI+): m/z 551 ([M+H]+), 573 ([M+Na]+), 389 ([M-Hexose+H]+); 1H NMR (400

210

MHz, DMSO-d6, COSY): δ [ppm] 3.11 [m, 2H, H-C(3´´), H-C(5´´)], 3.24 [m, 2H, H-

211

C(2´´), H-C(4´´)], 3.38 [m, 1H, H-C(6´´)], 3.57 [d, 1H, J=10.0 Hz, H-C(6´´)], 3.77 [s,

212

3H, H-C(7a)], 3.83 [s, 3H, H-C(5a)], 3.84 [s, 3H, H-C(3á)], 3.85 [s, 3H, H-C(4á)],

213

3.94 [s, 3H, H-C(6a)], 5.51 [d, 1H, J=7.4 Hz, H-C(1´´)], 7.11 [d, 1H, J=8.5, H-C(5´)],

214

7.19 [s, 1H, H-C(8)], 7.66 [dd, 1H, J=2.0, 8.5 Hz, H-C(6´)], 7.94 [d, 1H, J=2.0 Hz, H-

215

C(2´)];

216

57.2 [C(6a)], 61.1 [C(6´´)], 61.4 [C(7a)], 62.4 [C(5a)], 70.2-78.2 [C(3´´), C(5´´)], 74.7

217

[C(2´´)], 77.3 [C(4´´)], 97.8 [C(8)], 101.2 [C(1´´)], 111.7 [C(5´´)], 112.6 [C(10)], 113.2

218

[C(2´)], 122.2 [C(6´)], 137.1 [C(3)], 140.1 [C(6)], 149.0 [C(3´)], 150.0 [C(4´)], 153.2

219

[C(5)], 157.9 [C(7)], 172.4 [C(4)].

13

C NMR (100 MHz, DMSO-d6, HMQC, HMBC): δ [ppm] 55.9 [C(3á), C(4á)],

220

Identification of the Carbohydrate Moiety in Glycosides. An aliquot (200-

221

500 µg) of each purified glycoside was taken up in water (500 µL), hydrochloric acid

222

(4.0 mol/L; 500 µL) was added, and the mixture was heated at 110° C. After 1 h, the

223

solution was diluted with water (500 mL) and analysed for monosaccharides by

224

means of high-performance ion chromatography (HPIC) as reported earlier.35

225

Isolation of Limonoides (1, 2, 22) and Their Glucosides (3, 16-21) from

226

Orange Seeds. Orange seeds (100 g) were washed with water (2 x 150 mL) and



227

acetone (2 x 150 mL), frozen in liquid nitrogen, ground in a laboratory mill, and then

228

refluxed with methanol (2 x 300 mL) for 4 h at 60° C. The organic layer was filtered,

229

the solvent was removed in vacuum to obtain a crude isolate, which was taken up in

230

water (10 mL) and, then, applied onto the top of a water-cooled 300 x 35 mm i.d.

231

glass column filled with an aqueous slurry of Amberlite XAD-2 (BDH Chemicals Ltd,

232

Poole, England). Chromatography was performed with a flow rate of 5 mL/min using

233

the following sequence of solvents: water (200 mL; XAD fraction I), methanol/water

234

mixtures 20/80 (v/v, 200 mL; XAD fraction II), 40/60 (v/v, 200 mL; XAD fraction III),

235

60/40 (v/v, 200 mL, fraction IV, 80/20 (v/v, 200 mL, XAD fraction V), followed by

236

methanol (200 mL; XAD fraction VI). The individual XAD fractions I-VI were

237

separated from solvent in vacuum, followed by freeze-drying. XAD fractions I-IV and

238

V/VI, respectively, were dissolved in a 20/80 and 90/10 (v/v) mixture of methanol and

239

aqueous formic acid (0.1% in water), and, after membrane filtration, aliquots (250 µL)

240

were fractionated by means of semi-preparative HPLC on a 250 x 10 mm i.d., 5 µm,

241

Microsorb – MV RP-18 column (Varian, Germany). Monitoring the effluent by means

242

of UV detection (220 nm) and evaporative light scattering detection (ELSD), the XAD

243

fractions I-IV were separated at a flow rate of 3.5 mL/min with a gradient system

244

starting with a mixture (10/90, v/v) of acetonitrile/0.1% aqueous formic acid,

245

increasing the acetonitrile content to 20% within 15 min, holding for 10 min,

246

increasing to 30% within 15 min and, then, to 100% within another 5 min, thereafter

247

keeping the mobile phase constant for 5 min. A total of seven peaks were collected

248

into ice-cooled flasks. To gently remove formic acid, the collected fractions were put

249

on top of Strata C 18-E cartridges (10 g, 60 mL, 55 µm, Giga Tubes, Phenomenex,

250

Germany) pre-conditioned with water. After flushing the cartridges with water (150

251

mL), the cartridges were dried by a stream of nitrogen and, then, the target

252

compounds were eluted with methanol. The solvent was separated under vacuum,


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253

the residue of each fraction was taken up with water (5 mL) and freeze-dried twice to

254

afford limonin-17-O-β-D-glucopyranoside (3, Figure 1), deacetylnomilinic acid 17-O-

255

β-D-glucopyranoside (16), epiisoobacunoic acid 17-O-β-D-glucopyranoside (17),

256

methyldeacetylnomilinate

257

glucopyranoside

258

opbacunone 17-O-β-D-glucopyranoside (21) as white amorphous powders in a purity

259

of >99 % (HPLC/ELSD, HPLC/MS, 1H NMR).

(19),

17-O-β-D-glucopyranoside nomilinic

acid

(18),

nomilin

17-O-β-D-glucopyranoside

17-O-β-D(20),

and

260

XAD fractions V and IV were combined and separated using the same

261

chromatographic system, but another gradient system operated with a flow rate of

262

3.5 mL/min: starting with a mixture (30/70, v/v) of acetonitrile/0.1% aqueous formic

263

acid, the acetonitrile content was increased to 40% within 15 min, then to 80% within

264

10 min, and, finally, to 100% within additional 10 min. The effluent of three peaks

265

were collected and separated from solvent in vacuum to give limonin (1), nomilin (2),

266

and deacetylnomilin (22) in a purity of >99% (HPLC/ELSD, HPLC/MS, 1H NMR).

267

Sensory Analyses. General Conditions, Panel Training. Twelve subjects (six

268

women and six men, age 25-35 years), who have given the informed consent to

269

participate the sensory tests of the present investigation and have no history of

270

known taste disorders, participated for at least two years in weekly training sessions

271

to recognize the taste of aqueous solutions of reference compounds using nose-clips

272

as reported earlier.27,28,36-39 Prior to sensory analysis, the fractions or isolated

273

compounds were confirmed to be essentially free of solvents and buffer compounds

274

used as reported earlier.27,28 In order to minimize the uptake of any compound to the

275

best of our knowledge, all the sensory analyses were performed by using the sip-

276

and-spit method, that means the test materials were not swallowed but expectorated.

277

Taste Profile Analysis. Whilst wearing nose-clips, aliquots (5 mL) of orange

278

juice and, after 1+1 dilution with water, orange molasses were presented to the



279

sensory panelists who were asked to briefly swirl around the sample in the mouth for

280

5 s and, then, to expectorate. Using this sip-and-spit method, the panelists were

281

asked to score the taste qualities astringent, bitter, sour, and sweet on a scale from 0

282

(not detectable) to 5.0 (strong taste impression) (Table 1).

283

Taste Dilution Analysis (TDA). HPLC fractions were dissolved in bottled water

284

(8 mL; pH 4.5) in their “natural” concentration ratio, that means in the ratios as they

285

were obtained from orange juice, and, then, stepwise diluted 1+1 with bottled water

286

(pH 4.5). The serial dilutions of each fraction were presented in order of ascending

287

concentrations to the trained sensory panellists who were asked to evaluate the taste

288

impressions and to determine the dilution at which a taste difference between the

289

diluted extract and the blank (control) could just be detected by means of a half-

290

tongue-test. These so-called taste dilution (TD)-factors40 evaluated by four different

291

assessors in three different sessions did not differ by more than plus/minus one

292

dilution step and were averaged (Table 2).

293

Recognition Threshold Concentrations. Bitter recognition thresholds were

294

determined using a whole mouth sip-and-spit approach based on an ascending

295

three-alternative forced-choice procedure with bottled water (pH 4.5) as the solvent

296

as reported earlier.27,37,39 Threshold concentrations of astringent compounds were

297

determined in bottled water (pH 4.5) by means of the so-called half-tongue test using

298

ascending 1+1 dilutions of the samples following the procedure reported earlier.36

299

The threshold value of the sensory group was approximated for each compound by

300

averaging the threshold values of the individuals (geometric mean) in three

301

independent sessions (Table 3). Values between individuals and separate sessions

302

did not differ more than plus or minus one dilution step; that is, a threshold value of

303

24 µmol/L for 4 represents a range of 12-48 µmol/L.


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304

Taste Re-Engineering Experiments. To prepare basic taste recombinants

305

(expt. 1), hesperidin, carbohydrates, and organic acids summarized in groups III-V

306

(Table 4) were dissolved in their “natural” concentrations in bottled water and the pH-

307

value of this solution was adjusted to 3.5 by the addition of trace amounts of an

308

aqueous formic acid solution (1.0 mol/L). Towards stepwise preparation of the total

309

taste recombinant, the basic taste recombinant solution was spiked with the limonoid

310

glucosides 3, 16, 17, and 19-21 (expt. 2), with limonoid glucosides 3, 16, 17, and 19-

311

21 and limonoid aglycons 1 and 2 (expt. 3), with limonoid glucosides 13, 16, 17, and

312

19-21 and polymethoxylated flavones 4-10, 12, 14, and 15 (expt. 4), and with

313

limonoid aglycons 1 and 2, limonoid glucosides 3, 16, 17,and 19-21, as well as the

314

polymethoxylated flavones 4-10, 12, 14, and 15 (expt. 5). Each taste recombinant

315

was sensorialy evaluated and compared to authentic orange juice by means of taste

316

profile analysis.

317

Dose/Response Recordings. Serial 1:1 dilutions of (i) limonoid aglycons (1, 2),

318

(ii) glucoside 3, (iii) polymethoxylated flavones (4-8, 10, 11, 12, and 15), (iv) mixtures

319

of limonin (1) and polymethoxylated flavones (4-7, 9, 10, 12, and 15; each in its

320

“natural” concentration ratios as given in Table 4; 0.25, 0.50, and 0.75 mg/100 g),

321

and (v) mixtures of nomilin (2) and polymethoxylated flavones (4-7, 9, 10, 12, and 15;

322

in their concentration ratio as present in orange juice; Table 4; 0.50 mg/100 g) were

323

used to perform human dose-response recordings following the procedure reported

324

earlier.38,39 Using a half-tongue tasting procedure, the solutions of the individual

325

compounds were applied in binary combinations to one side of the tongue and

326

panelists were asked to determine which side showed the stronger sensation. In

327

order to assess reliable taste intensities of compounds, the taste intensity of the

328

individual compounds were compared on a five-point scale (0.25 scale units) to

329

reference solutions containing caffeine in concentrations of 0.75 (0), 1.5 (0.25), 3.0



330

(0.5), 6.0 (1.0), 12 (2.25), 24 (3.0), 48 (3.5), and 96 mmol/L (4.5) with pre-defined

331

bitter intensities given in parenthesis. After the taste intensity of each compound at its

332

maximum solubility had been rated, the taste intensities of the other dilutions were

333

determined by using the half-tongue tasting method so that one dilution of an

334

individual compound was rated against the intensity of another dilution of the same

335

compound, and by cross-checking the taste intensity between the different

336

compounds at the same dilution. The dose/response functions of three individual

337

sessions were averaged. Human response functions with dose-over-threshold (DoT)-

338

factors on the x-axis and taste intensities on the y-axis were recorded for each

339

individual subject in triplicates.

340

Bitterness Enhancement of Limonin Solutions by Polymethoxylated Flavones.

341

Solutions of limonin (16 µmol/L) spiked with individual flavones (4-8, 10, 11, 12, and

342

15; 0.5 mg/100 g) were presented to the sensory panelists who were asked to rate

343

the bitter taste intensity against a reference solution containing limonin (16.0 µmol/L).

344

The sensory data were determined as the mean of triplicates.

345

Sensory Interactions between Sugars, Limonoids, and Polymethoxylated

346

Flavones. A matrix A of 81 test samples was prepared with varying levels (6.1, 6.6,

347

7.1, 7.6, 8.1, 8.6, 9.1, 9.6, 10.1 g/100 g) of a glucose/fructose/sucrose mixture (molar

348

ratio 1.02:1.23:1.18 as found for OJ1, Table 4) and varying concentrations (0.25,

349

0.55, 0.65, 0.75, 0.85, 1.25, 1.65, 2.05, 2.45, and 2.85 mg/100 g) of

350

polymethoxylated flavones (4-10, 12, 14, and 15, in “natural” concentration ratios as

351

given for OJ1 in Table 4) while the levels of a limonin/nomilin mixture (molar ratio

352

1:2; 0.6 mg/100g), citric acid (640 mg/g), and malic acid (170 mg/100 g) were kept

353

constant. In addition, two additional sets of 56 test samples (matrices B and C) were

354

prepared with varying levels of the glucose/fructose/sucrose mixture detailed above

355

and varying concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg/100 g) of a


Page 14 of 58

Page 15 of 58


356

limonin/nomilin mixture (molar ratio 1:2), while the concentration of citric acid (640

357

mg/g) and malic acid (170 mg/100 g) was kept constant and that of flavones adjusted

358

to 1.1 (matrix B) or 0.6 mg/100 g (matrix C). Randomly selected samples out of the

359

matrices A and B were presented to the trained sensory panelists who were asked to

360

judge the bitter taste intensity of each test solution on a scale from 0 (no taste

361

observed) to 10 (extremely intense bitterness) by comparing it with an ascending

362

dilution series of limonin as the reference. To avoid fatigue of the panelists, a

363

maximum of eight solutions per day were presented to the sensory panel. The data

364

were calculated as the means of duplicates, intensity values between trained

365

individuals and separate sessions did not differ more than plus or minus 0.4 units.

366

Quantitative Analysis. Polymethoxylated Flavones. For quantitative analysis

367

of compounds 4-13, an aliquot of orange juice (15 g) was spiked with an internal

368

standard solution (1 mL) of 4′-methoxyflavone (16 mg) in methanol/water (80/20, v/v;

369

100 mL), diluted with water to about 25 mL, and equilibrated for 30 min upon

370

ultrasonification. After centrifugation (15 min, 1006 × g), an aliquot (10 mL) of the

371

supernatant was applied on top of a water-conditioned Strata C 18-E, 55 µm, RP-18-

372

cartridge (10 g, 60 mL, Giga Tubes, Phenomenex), followed by elution with water (30

373

mL). The target analytes were then eluted with methanol (40 mL), the solvent was

374

separated in vacuum, the residue was taken up in water/methanol (80/20, v/v; 1.0

375

mL), and an aliquot (20-30 µL) was analyzed by means of HPLC-DAD on a 125 x

376

4.6 mm i.d., 5 µm, Microsorb – MV 100-5C 18 column (Varian, Germany). Operating

377

with a flow rate of 1.0 mL/min and monitoring the effluent at 256 nm, chromatography

378

was performed by means of a solvent gradient using 0.03 % aqueous formic acid

379

(solvent A) and acetonitrile (solvent B): 0 min (A/B, 80/20, v/v) - 15 min (A/B, 70/30,

380

v/v) - 52 min (A/B, 62/38, v/v) - 60 min (A/B, 40/60, v/v) - 65 min (A/B, 40/60, v/v) - 67

381

min (A/B, 0/100, v/v). Quantitation was performed by means of HPLC-DAD with



382

external 5-point calibration using standard solutions of polymethoxylated flavones in

383

concentrations between 0.1 and 30.0 mg/L.

384

For the quantitative analysis of the flavone glucosides 14 and 15, orange juice

385

(25 g) was made up to 50 mL with methanol, ultrasonificated for 20 min, centrifuged

386

(15 min, 1006 × g), membrane filtered, and, then, an aliquot (5 µL) of the supernatant

387

was analyzed by means of HPLC-MS/MS in the ESI+ mode on a 150 x 2 mm, 4 µm,

388

Synergi Fusion column (Phenomenex) equipped with a 4 x 2 mm pre-column of the

389

same type operated at a flow rate of 250 µL/min. Using aqueous formic acid (0.1 %)

390

as solvent A and acetonitrile as solvent B, chromatography was done as follows: 0

391

min (A/B, 60/40, v/v) – 10 min (A/B, 40/60, v/v) – 12 min (A/B, 40/60, v/v) – 15 min

392

(A/B, 0/100, v/v). Compounds 14 (m/z 551.3→389.2; +90/+30/+15) and 15 (m/z

393

581.3→419.2; +90/+30/+15) were analyzed in the positive electrospray ionization

394

mode (ESI+) using the mass transitions and declustering potential (DP, in V), collision

395

energy (CE, in V), and cell exit potential (CXP, in V) given in parentheses. Analysis

396

was performed with external 5-point-standard calibration with standard solutions of

397

14 and 15 in concentrations between 0.70 and 14.7 mg/L.

398

Limonoid Aglycones 1 and 2. Orange juice (20 g) was made up to 50 mL with

399

methanol, ultrasonificated for 20 min, centrifuged (10 min, 1006 × g), membrane

400

filtered, and, then, an aliquot (5 µL) was analyzed by means of HPLC-MS/MS in the

401

ESI+-mode on a 150 x 2 mm, 4 µm, Synergi Fusion column (Phenomenex) equipped

402

with a 4x2 mm pre-column of the same type operated at a flow rate of 250 µL/min.

403

Using aqueous formic acid (0.1 %) as solvent A and acetonitrile as solvent B,

404

chromatography was done as follows: 0 min (A/B, 85/15, v/v) – 5 min (A/B, 55/45,

405

v/v) – 15 min (A/B, 30/70, v/v) – 18 min (A/B, 0/100, v/v). Compounds 1 (m/z

406

471.0→425.0; +40/+30/+15) and 2 (m/z 515.0→469.0; +70/+30/+15) were analyzed

407

in the ESI+ mode using the mass transitions and declustering potential (DP, in V),


Page 16 of 58

Page 17 of 58


408

collision energy (CE, in V), and cell exit potential (CXP, in V) given in parentheses.

409

Analysis was performed with external 6-point-standard calibration with standard

410

solutions of 1 and 2 in concentrations between 0.3 and 12.0 mg/L.

411

Limonoid Glucosides 3, 16, 17, 19-21. Orange juice (20 g) was made up to 100

412

mL with methanol, ultrasonificated for 20 min, 1:20 diluted with water, centrifuged (10

413

min, 3000 rpm), membrane filtered, and, then, an aliquot (5 µL) of the supernatant

414

was analyzed by means of HPLC-MS/MS in the ESI--mode on a 150x2 mm, 4 µm,

415

Synergi Fusion column (Phenomenex) equipped with a 4x2 mm pre-column of the

416

same type operated at a flow rate of 250 µL/min. Using aqueous formic acid (0.1 %)

417

as solvent A and acetonitrile as solvent B, chromatography was done as follows: 0

418

min (A/B, 60/40, v/v) – 10 min (A/B, 45/55, v/v) – 13 min (A/B, 0/100, v/v) – 18 min

419

(A/B, 0/100, v/v). The target compounds were analyzed in the negative electrospray

420

ionization mode (ESI-) using the mass transitions and declustering potential (DP, in

421

V), collision energy (CE, in V), and cell exit potential (CXP, in V) given in

422

parentheses: 3 (m/z 649.0→605.0; -70/-30/-15), 16 (m/z 669.0→609.0; -50/-40/-15),

423

17 (m/z 651.0→591.0; -60/-40/-15), 19 (m/z 693.0→59.0; -80/-102/-15), 20 (m/z

424

711.0→59.0; -65/-100/-15), 21 (m/z 633.0→59.0; -80/-86/-15). Analysis was

425

performed with external 6-point-standard calibration with standard solutions of each

426

limonoid glucoside in concentrations between 0.06 and 4.2 mg/L.

427

Hesperidin (23). Orange juice (10 g) was centrifuged (15 min, 1006 × g), the

428

residue was suspended in an aqueous ammonium oxalate solution (0.025 mol/L; 10

429

mL), followed by centrifugation, and was then extracted with dimethyl formamide (10

430

mL), followed by centrifugation. The combined supernatants were made up to 50 mL

431

with aqueous formic acid (0.01 mol/L in water) and an aliquot (20 µL) was analyzed

432

by means of HPLC-DAD on a 125 x 4.6 mm i.d., 5 µm, Microsorb – MV 100-5C 18

433

column (Varian, Germany). Operating with a flow rate of 1.0 mL/min and monitoring



434

the effluent at 280 nm, chromatography was performed isocratically with a mixture

435

(79/21, v/v) of 0.1 % aqueous formic acid and acetonitrile. Quantitation was

436

performed by means of HPLC-DAD with external 5-point-calibration using hesperidin

437

standard solutions in concentrations between 20.0 and 140.0 mg/L.

438

Organic Acids and Carbohydrates. Organic acids and carbohydrates were

439

quantitated by means of high-performance ion chromatography (HPIC) on a ICS-

440

2500 ion chromatography system (Dionex, Idstein, Germany) equipped with a 250 x

441

2 mm IonPac AS11-HC column (Dionex) and a 250 x 2 mm CarboPac PA-10 column

442

(Dionex), respectively, following the protocols reported earlier.35

443

High Performance Liquid Chromatography (HPLC). For isolation of

444

polymethoxylated flavones, the HPLC apparatus (Jasco, Groß-Umstadt, Germany)

445

consisted of two pumps (PU 2086/2087), a gradient mixer (1000 µL), a Rheodyne

446

injector (250 or 300 µL loop), and a multiwavelength detector (MD 2010plus, Jasco,

447

Germany) monitoring the effluent in a wavelength range between 220 and 500 nm.

448

For isolation of limonoides and their glucosides, the HPLC apparatus consisted of

449

two pumps (Sykam, S 1122), a gradient mixer (Sunchrom, dynamic/statistic gradient

450

mixer), a Rheodyne injector (250 µL loop), an autosampler (Spark, Midas 380), a

451

diode array detector (Sunchrom, SpectraFlow 600 DAD), monitoring the effluent in a

452

wavelength range between 200 and 600 nm, a splitting module (Upchurch, P-470

453

graduated microsplitter), and a type Sedex 85 LT-ELSD evaporative light scattering

454

detector (S.E.D.E.R.E.) equipped with a nebulizer (p = 2,6 bar, T = 40 °C, 2.5-

455

200 µL/min). For quantitative analysis of polymethoxylated flavones and hesperidine,

456

an analytical HPLC (Merck Hitachi, Eching) was equipped with two type L-7100a

457

pumps, a type L-7612a degasser, a type L-7200 autosampler, and a type L-7450

458

diode array detector (DAD).


Page 18 of 58

Page 19 of 58


459

Liquid Chromatography/Mass Spectrometry (LC/MS). Electrospray ion-

460

ization (ESI) spectra of target compounds were acquired on an API 4000 Q-Trap

461

LC/MS/MS system (AB Sciex Instruments, Darmstadt, Germany) with direct loop

462

injection of the sample (2-20 µL). For HPLC-MS/MS analyses the API 4000 Q-Trap

463

LC/MS/MS system was coupled to an Agilent 1100 HPLC system equipped with a

464

150 x 2 mm, 4 µm, Synergi Fusion column (Phenomenex) and a 4 x 2 mm pre-

465

column of the same type. Ionization was performed by means of electrospray

466

ionization (ESI). The spray voltage was set at –4500 V in the ESI--mode and at 5500

467

V in the ESI+-mode. Nitrogen served as curtain gas (20 psi), the declustering

468

potential was set at –10 to –30 V in the ESI- mode and 30-40 V in the ESI+-mode.

469

The mass spectrometer was operated in the full scan mode monitoring positive or

470

negative ions. Fragmentation of [M-H]- and [M+H]+ molecular ions into specific

471

product ions was induced by collision with nitrogen (4 x 10-5 torr) and a collision

472

energy of -20 to -70 V or 20 to 60 V.

473

Nuclear Magnetic Resonance Spectroscopy (NMR). Samples were dissolved

474

in DMSO-d6 or MeOD-d4 with tetramethylsilane as internal standard, placed into 178

475

x 5 mm i.d. NMR tubes (Schott Professional), and were then analyzed by means on a

476

DPX 400 NMR spectrometer (Bruker, Rheinstetten, Germany). Data processing was

477

performed by using the NMR Software Mestre-C.

478 479 480

RESULTS AND DISCUSSION

481 482

To identify the compounds responsible for the typical taste of orange juice,

483

orange juice OJ1, prepared from Valencia late oranges harvested in November 2003,



484

was presented to trained sensory panelists who were asked to rate the intensities of

485

the taste descriptors sweet, sour, bitter, and astringent on a linear scale from 0 (not

486

detectable) to 5 (strongly detectable). High scores were found for the intensities of

487

sourness (2.3) and sweetness (2.0), followed by bitterness and astringency judged

488

with somewhat lower intensities of 1.8 and 1.1, respectively (Table 1). To investigate

489

the hydrophobicity of the key taste molecules, juice OJ1 was separated by means of

490

sequential solvent fractionation.

491

Solvent Fractionation of Orange Juice OJ1. Orange juice was extracted

492

with solvents of increasing polarity to obtain the pentane soluble fraction I, the

493

dichloromethane soluble fraction II, the ethyl acetate soluble fraction III and the

494

remaining aqueous fraction IV, which were freed from solvent in high-vacuum, taken

495

up in water in their “natural” concentration and, after adjustment to pH 4.5, were

496

presented to the trained sensory panel for taste profile analysis (Table 1). Fraction IV

497

was judged with the highest scores for sweetness (2.5) and sourness (1.5), whereas

498

bitterness (0.5) and astringency (0.4) were only marginally detected. In comparison,

499

the taste profile of fraction II was dominated by an intense bitter taste (3.3)

500

accompanied by velvety-like astringent mouthfeel (1.1). Also fraction III exhibited

501

some pronounced bitterness (1.1) and astringency (1.0) with only low ratings for

502

sourness (0.9) and sweetness (0.1). In comparison, the most hydrophobic fraction I

503

showed only faint taste activity.

504

Although fraction II showed by far the highest impact for bitterness and

505

astringency, the yield of this fraction isolated from orange juice were too low to allow

506

straightforward structure elucidation of the key taste molecules by means of NMR

507

spectroscopy. As preliminary taste profile analyses revealed orange molasses, a

508

main by-product of the citrus industry, as a rich source for bitter and astringent

509

compounds judged with intensities of 4.1 and 3.1 (Table 1), orange molasses was


Page 20 of 58

Page 21 of 58


510

separated by means of sequential solvent extraction in order to obtain the candidate

511

bitter and astringent compounds in higher amounts. Matching well with the sensory

512

analysis of the fractions isolated from orange juice, fraction II isolated from orange

513

molasses showed high scores for bitterness (4.5) and astringency (2.4).

514

Comparison of the RP-HPLC chromatogram of fraction II isolated from orange

515

juice with that obtained from orange molasses demonstrated that the presence of the

516

same compounds in both bitter fractions, just differing in the quantity of the individual

517

molecules (data not shown). Due to the higher yield of fraction II from orange

518

molasses, the isolation and structure determination of bitter and astringent

519

compounds was performed using fraction II from orange molasses.

520

Identification of Sensometabolites in Fraction II. To sort out the strongly

521

taste-active compounds from the bulk of less taste-active or tasteless substances,

522

fraction II was separated by RP-HPLC to give 19 fractions (II/1 - II/19; Figure 3),

523

which were collected individually, freeze-dried, taken up in water in their “natural”

524

concentrations and, then, used for taste dilution analysis (Table 2). Although each

525

individual fraction exhibited some bitterness and astringency, fractions II/3, II/8-11,

526

II/14, and II/16 were judged with the highest TD-factors ranging from 128 to 512 for

527

velvety-like astringency. In addition, fractions II/8-12 and II/16 were evaluated as the

528

most intense bitter fractions showing bitter taste even in a dilution of 1:32 to 1:256,

529

respectively. Due to their taste impact, the following identification experiments were

530

targeted towards the key taste compounds in fractions II/3, II/4, and II/8-19.

531

HPLC-DAD, HPLC-ELSD, LC-MS, and NMR analysis, followed by co-

532

chromatography with reference materials led to the identification of the triterpene

533

lactones limonin (1) and nomilin (2) as key bitter compounds in HPLC fractions II/12

534

and II/17 and the polymethoxylated flavones 5,6,7,3′,4′-pentamethoxyflavone (4),

535

5,6,7,4′-tetramethoxyflavone

(5),

and

5,6,7,8,4′-pentamethoxyflavone


(6)

as


536

astringent and bitter tastants in fractions II/10, II/15, and II/18, respectively (Table 2).

537

Moreover, LC-MS/MS,

538

correlation experiments (HMQC, HMBC) revealed the unequivocal identification of

539

5,7,8,3′,4′-pentamethoxyflavone (7, fraction II/8), 3,5,7,8,3′,4′-hexamethoxyflavone

540

(8, fraction II/9), 3,5,6,7,3′,4′-hexamethoxyflavone (9, fraction II/1), 5,6,7,8,3′,4′-

541

hexamethoxyflavone (10, fraction II/14), 5,7,8,4′-tetramethoxyflavone (11, fraction

542

II/13), 3,5,6,7,8,3′,4′-heptamethoxyflavone (12, fraction II/16), and 4′-hydroxy-

543

5,6,7,8,3′-pentamethoxyflavone (13, fraction II/19), respectively. Although 4-13 were

544

identified earlier in literature,29-32 their bitter taste has not yet been reported.

1

H/13C NMR, homonuclear (COSY) and heteronuclear

545

Due to their low yield and impurity, tastants 14 and 15 isolated from fractions

546

II/3 and II/4 were enriched by means of flash-chromatography on RP-18 and gel

547

adsorption chromatography on Sephadex LH-20, and then purified by re-

548

chromatography (RP-HPLC). LC/MS analysis of the compound 14 revealed the

549

pseudomolecular ions m/z 551 ([M+H]+), 573 [M+Na]+, and 589 [M+K]+, thus

550

indicating a molecular weight of 550 Da. MS/MS fragmentation experiments with the

551

[M+H]+ ion demonstrated the cleavage of a hexose moiety by the loss of 162 amu.

552

The 1H NMR as well as the COSY spectrum indicated a total of 25 protons, four of

553

which resonated between 7.11 and 7.94 ppm as expected for aromatic protons and

554

five signals, each integrating for three protons, showed chemical shifts between 3.77

555

and 3.94 ppm as expected for methoxy groups. The remaining protons observed

556

between 3.11 and 3.57 ppm as well as at 5.51 ppm indicated the presence of a

557

hexose moiety linked at position 3 of the methoxyflavone skeleton. For final

558

identification of the hexose moiety, sugar analysis was performed in an acidic

559

hydrolysate of 14 by means of high-performance ion chromatography (HPIC).

560

Comparison of retention time and co-chromatography with a glucose reference

561

revealed glucose as the sugar moiety and led to the identification of 5,6,7,3′,4′-


Page 22 of 58

Page 23 of 58


562

pentamethoxyflavone-3-O-β-D-glucopyranoside (14, Figure 2), which to the best of

563

our knowledge has not been previously reported in literature. Rather similar NMR

564

data were obtained from compound 15, just lacking one aromatic proton and

565

exhibiting an additional methoxy group at position 8. Acidic hydrolysis, followed by

566

HPIC analysis, as well as comparison of spectroscopic data with those reported in

567

literature34

568

hexamethoxyflavone-3-O-β-D-glucopyranoside (15).

revealed the astringent and bitter tastant 15 as

5,6,7,8,3′,4′-

569

Isolation of Limonoids and their Glucosides. Compared to fraction II, the

570

bitter and astringent tasting compounds in orange juice fraction III were expected to

571

be more hydrophilic, but their low amounts did not allow a straightforward structure

572

elucidation. As preliminary LC-MS screening experiments located limonin-17-O-ß-D-

573

glucopyranoside in fraction III, the following experiments were targeted towards the

574

isolation of a series of limonoid glycosides from orange seeds as these are known as

575

a rich source of limonoid glucosides.41,42 Once isolated, these glucosides should be

576

used as reference materials for sensory analysis and as external standards for their

577

accurate quantitative analysis in orange juice.

578

A methanol extract prepared from freshly ground orange seeds was separated

579

on an XAD resin, followed by RP-HPLC purification to afford a total of three limonoid

580

aglycones and seven glucosides (Figure 4). By means of HPLC-MS/MS and 1D/2D-

581

NMR experiments and comparison with literature data, these bitter and astringent

582

compounds were identified as limonin 17-ß-D-glucopyranoside, 3,17 deacetylnomilinic

583

acid

584

glucopyranoside, 17,43 methyldeacetylnomilinic acid 17-O-ß-D-glucopyranoside, 18,21

585

nomilin-17-O-ß-D-glucopyranoside, 19,20 nomilinic acid 17-O-ß-D-glucopyranoside,

586

20,43 obacunon 17-O-ß-D-glucopyranoside, 21,20 deacetylnomilin, 22,21 limonin (1),

587

and nomilin (2), respectively (Figure 1).

17-O-ß-D-glucopyranoside,

16,43

epiisoobacunoic


acid

17-O-ß-D-


588

Identification of Sensometabolites in Orange Juice Fractions III and IV.

589

RP-HPLC/DAD and RP-HPLC-MS/MS analysis, followed by co-chromatography with

590

the reference compounds isolated from orange seeds and molasses, respectively,

591

led to the identification of the limonoid aglycones 1 and 2, the limonoid glucosides 3,

592

16, 17, and 19 - 21, and low amounts of the polymethoxylated flavones 4-10, 12, 14,

593

and 15 in the astringent and bitter tasting orange juice fraction III. In addition,

594

degustation experiments revealed an intense astringent orosensation imparted by the

595

major peak detected at 280 nm. By comparison of chromatographic and LC-MS/MS

596

data with those reported in the literature,44 followed by co-chromatography with the

597

reference compound, this astringent compound was identified as hesperidin (23).

598

Sensory analysis revealed a recognition threshold of 11 µmol/L for the astringent

599

sensation induced by this flavanone glycoside.

600

In order to analyze the major compounds imparting the sour and sweet taste

601

impression induced by juice fraction IV, organic acids and carbohydrates were

602

analyzed by means of HPIC. Citric acid, malic acid, and isocitric acid were identified

603

as major organic acids and glucose, fructose, and sucrose as predominating

604

carbohydrates.

605

Sensory Activity of Astringent and Bitter Sensometabolites. After

606

checking the purity of all compounds (HPLC-MS, 1H NMR), recognition threshold

607

concentrations for bitter taste and the astringent oral sensation were determined in

608

aqueous solutions by means of a three-alternative forced choice test and a half-

609

tongue test, respectively (Table 3). The polymethoxylated flavones 4-15 imparted a

610

velvety-like astringency to the oral cavity at very low threshold concentrations ranging

611

from 4 to 51 µmol/L. With the exception of 3,5,7,8,3′,4′-hexamethoxyflavone (8) and

612

5,7,8,4′-tetramethoxyflavone (11), all the other flavones exhibited also a long-lasting

613

bitterness with recognition thresholds between 19 and 103 µmol/L depending on the


Page 24 of 58

Page 25 of 58


614

chemical structure. Structure/activity considerations indicated an influence of the

615

methoxylation pattern on the bitterness of the flavones. Tetramethoxylation of the B-

616

ring was found to slightly increase thresholds when compared to structures

617

decorated with methoxy groups only at positions 5, 6, and 7. For example,

618

compounds 4 and 5 exhibiting three methoxygroups at the B ring imparted somewhat

619

lower bitter thresholds of 56 and 44 µmol/L, respectively, when compared to 10 (103

620

µmol/L) and 6 (93 µmol/L) just differing by one additional methoxy group at position 8

621

(Figure 2). Whereas the methoxylation pattern of the C-ring did not seem to

622

influence the bitter threshold, the substitution of one methoxy group by a hydroxyl

623

function induced a decrease of the bitter threshold concentration, e.g. the bitter

624

threshold

625

hexamethoxyflavone 10. Also position 3 was found to play an important role in the

626

perceived bitterness of these flavones. The threshold of 19 and 24 µmol/L found for 9

627

and 12 showed that methoxylation at position 3 led to lower thresholds compared to

628

glucosylation as found in 14 and 15 exhibiting thresholds of 61 and 78 µmol/L,

629

respectively.

of

13

was

found

to

be

4

times

lower

when

compared

to

630

Among the limonoid aglycones (Table 3), limonin (1) and nomilin (2) were

631

found to induce bitterness at rather low recognition thresholds of 4 and 13 µmol/L,

632

thus confirming literature data.9,10 In comparison, deacetylnomilin (22) did not show

633

any bitter taste up to the maximum test concentration of 106 µmol/L, but imparted an

634

intense astringent and mouth-drying orosensation above 13 µmol/L. The limonoid

635

glucosides showed a silky astringent orosensation with low threshold concentrations

636

between 5 and 27 µmol/L for compounds 3, 16, 17, and 21, and somewhat higher

637

astringency thresholds of 205 and 404 µmol/L for compounds 20 and 19, respectively

638

(Table 3). In addition, the sensory panel reported compounds 3, 16, 17, and 21 to

639

induce a distinct bitter taste perceived at the posterior tongue.



640

Quantitation of Sensometabolites in Orange Juices. After identification of

641

the non-volatile sensometabolites and determination of their taste threshold

642

concentrations, polymethoxylated flavones were analyzed by means of HPLC/DAD,

643

limonoid aglycones, limonoid glucosides, as well as the polymethoxylated flavone

644

glucosides by means of HPLC-MS/MS, and carbohydrates and organic acids by

645

means of HPIC in the orange juices OJ1 and OJ2 prepared from Valencia oranges

646

harvested in November 2003 and March 2006, respectively.

647

The limonoid aglycones 1 and 2, summarized in group I in Table 4, were

648

found in concentrations of 13.1 and 3.8 µmol/L in OJ1, whereas OJ2 contained

649

comparatively low amounts of 2.1 and 0.1 µmol/L, respectively. To evaluate the taste

650

impact of these sensometabolites, the dose-over-threshold (DoT)-factors were

651

calculated as the ratio of the concentration to the taste threshold of a compound.

652

Only limonin (1) exceeded its threshold concentration in OJ1 and was evaluated with

653

a DoT-factor of 3.3, followed by nomilin (2) with a DoT-factor of 0.3 only (Table 4). In

654

comparison, none of these limonoids exceeded their threshold in OJ2.

655

The astringent and bitter tasting limonoid glucosides 3, 16, 17, and 19-21 as

656

well as the polymethoxylated flavones 4-10, 12, 14, and 15 were summarized in

657

tastant group II (Table 4). The limonoid glucosides were found in both juices in high

658

concentrations, e.g. 139.2 or 113.9 µmol/L of 3 and 93.7 or 58.5 µmol/L of 20 was

659

found in OJ1 or OJ2, respectively. A comparatively low concentration of 4.8 µmol/L

660

was determined for glucoside 21. Calculation of DoT-factors for the astringent

661

orosensation revealed a value of 8.9 and 7.2 for 3 in OJ1 and OJ2, respectively

662

(Table 4). Interestingly, glucoside 3 even exceeded its threshold concentration for

663

bitterness by a factor of 1.3 and 1.1, respectively, thus given a first evidence for its

664

contribution to the bitter taste of orange juices. The concentration of flavones 4-10,

665

12, 14, and 15 ranged between 0.02 and 5.8 µmol/L for OJ1 and between 0.03 and


Page 26 of 58

Page 27 of 58


666

10.8 µmol/L for OJ2, respectively. Compound 10 was identified as the major flavone

667

in both juices, followed by compound 12 with 4.6 and 6.6 µmol/L and by compound 4

668

with 3.0 and 10.3 µmol/L in OJ1 and OJ2, respectively. Calculation of DoT-factors

669

revealed that exclusively 3,5,6,7,8,3′,4′-heptamethoxyflavone (12) reached its

670

astringency threshold in both orange juices. As none of the polymethoxylated

671

flavones were found in supra-threshold concentrations for bitter taste, the individual

672

derivatives cannot be considered as primary taste contributors to bitter taste of

673

orange juice. However, recent cell-based experiments demonstrated that structurally

674

related bitter compounds do additively co-activate bitter receptors.45 Therefore, the

675

structural similarity of the group of polymethoxylated flavones might enable an

676

additive taste contribution of 4-10, 12, 14, and 15, respectively.

677

High concentrations of 650.8 and 752.5 µmol/L were found for the velvety

678

astringent hesperidin (23) in OJ1 and OJ2, respectively (Table 4). Calculation of the

679

DoT-factors for astringency revealed a value of 59.2 and 68.4, respectively, thus

680

implying 23 as a key contributor to the astringency of orange juices.

681

Among the carbohydrates (group IV, Table 4), sucrose, fructose, and glucose

682

were found almost in equimolar comcentrations. Calculation of DoT-factors indicated

683

sucrose (4.6 and 3.6) and fructose (2.2 and 1.6) as the key sweet contributors in OJ1

684

and OJ2, respectively, whereas glucose just reached its threshold concentrations.

685

In tastant group V, comprising the organic acids, high concentrations of

686

33.9/31.8 and 11.2/17.2 mmol/L were found for citric acid and malic acid in OJ1/2,

687

respectively (Table 4). Both acids exceeded their individual taste threshold in both

688

orange juices, whereas the minor isocitric acid was present in subthreshold

689

concentrations.

690

Re-Engineering the Sensometabolome of Orange Juices OJ1 and OJ2. In

691

order to functionally validate the analytical data obtained, taste re-engineering



692

experiments were performed. First, a cocktail of hesperidin, carbohydrates, and

693

organic acids, each in its “natural” concentration (Table 4), was dissolved in bottled

694

water and the pH was adjusted to 3.5. Aliquots of this basic taste recombinant

695

solution were then spiked with the limonoid glucosides 3, 16, 17, and 19-21 (expt. 2),

696

with limonoid glucosides 3, 16, 17,and 19-21 and limonoid aglycons 1 and 2 (expt. 3),

697

with limonoid glucosides 3, 16, 17,and 19-21 and polymethoxylated flavones 4-10,

698

12, 14, and 15 (expt. 4), and with limonoid aglycons 1, 2, limonoid glucosides 3, 16,

699

17, 19-21, and polymethoxylated flavones 4-10, 12, 14, 15 (expt. 5.). The

700

recombinant solutions prepared in expts. 1-5 were then sensorially evaluated and

701

compared to authentic orange juices OJ1 and OJ2, respectively, by means of taste

702

profile analysis (Figure 5).

703

Although a triangle test showed a significantly different overall taste profile of

704

OJ1 and OJ2 (p = 0.01), both authentic juices were judged with rather similar

705

intensities for astringency and bitterness with scores of about 1.1 (OJ1) and 1.8

706

(OJ2), respectively (Figure 5, A). It is however interesting to notice, that the

707

bitterness of OJ1 was perceived on the entire tongue, whereas the bitter taste of OJ2

708

was located mainly at the posterior part of the tongue and was perceived as more

709

long-lasting. OJ1 was rated with higher scores for sourness (2.9) than OJ1 (2.3),

710

which was perceived as more sweet (2.0) when compared to OJ2 (1.7).

711

Sensory analysis of the basic taste recombinants containing organic acids,

712

sugars, and hesperidin demonstrated a good match with the authentic juices OJ1

713

and OJ2 with regard to sour taste, whereas sweetness was evaluated with slightly

714

higher scores in the recombinants (expt. 1; Figure 5, B). Surprisingly, astringency

715

was judged with rather low intensities of 0.3 and 0.4, respectively, although the

716

astringent hesperidin was present in both tastant cocktails.

717

Addition of limonoid glucosides 3, 16, 17, and 19-21 (expt. 2) to the basic


Page 28 of 58

Page 29 of 58


718

recombinant solutions did not show any significant influence on sweetness and

719

sourness. However, astringency and bitterness intensities increased to 0.8/0.6 and

720

0.7/0.4 for OJ1/2, although the taste intensities of the authentic juices were not yet

721

reached (Figure 5, C). Interestingly, all members of the sensory panel reported that

722

the recombinant containing the limonoid glucosides were perceived as “more rich”,

723

“more complex”, and “more full-bodied”. These sensory data nicely confirm the

724

proposed sensory contribution of these glucosides and, in particular, of compound 3

725

on the basis of calculated DoT-factors (Table 4).

726

In expt. 3, the basic recombinant was spiked with limonoid glucosides and

727

limonoid aglycons. Sensory analysis revealed a significant increase of bitterness

728

from 0.7 (Figure 5, B) to 1.5 (Figure 5, D) for OJ1 whereas the bitter taste intensity

729

of the recombinant of OJ2 was only marginally increased from 0.4 to 0.7. This is well

730

in line with the high DoT-factor calculated for limonin (1) in OJ1 when compared to

731

OJ2. Compared to expt. 2 (Figure 5, C), the bitterness perceived for the partial OJ1

732

recombinant in the presence of the limonoid aglycones 1 and 2 was described as

733

unpleasant and biting bitter and, in addition, the intensity of the sweetness was

734

slightly suppressed (Figure 5, D). This confirms the previous finding that

735

concentrations of 1 above 6 ppm will reduce the acceptability of orange juices.46

736

Neither astringency, nor sourness was affected significantly by the presence of these

737

triterpenoids.

738

In expt. 4, the taste recombinant containing the limonoid glucosides were

739

spiked with the polymethoxylated flavones instead of limonoid aglycons (expt. 3).

740

When compared to expt. 2 (Figure 5, C), the astringency of the recombinants

741

increased to 1.0 and 1.1, respectively, thus matching the astringency scores of the

742

authentic juices OJ1 and OJ2 (Figure 5, E). In addition, a significant increase of

743

bitterness (0.4→1.4) was observed for the partial recombinant of OJ2, whereas the



744

influence of bitter taste for the OJ1 sample was only marginal. This is well in line with

745

the higher content of 4-10, 12, 14, and 15 in OJ2 (Table 4) and, for the first time,

746

unequivocally demonstrates the contribution of these polymethoxylated flavones to

747

the astringency as well as the bitter taste of orange juice. Sensory analysis of total

748

recombinants, including all taste compounds summarized in Table 4 (expt. 5),

749

revealed an excellent match between the authentic juices OJ1 and OJ2 and their

750

corresponding taste recombinants (Figure 5, F), thus confirming that the key

751

sensometabolites of orange juice were successfully identified and quantified.

752

It is interesting to notice that, although the taste profiles of OJ1 and OJ2 are

753

almost identical (Figure 5, A), the quantitative composition of the sensometabolome

754

in both juices is very different (Table 4). This clearly indicates that not a single

755

compound such as, e.g. limonin (1), can be considered a suitable marker compound

756

to analytically measure bitterness. More important, the taste percept of orange juice

757

seems to be created by a rather complex interplay of limonin, limonoid glucosides,

758

polymethoxylated flavones, organic acids, and sugars and, therefore, the prediction

759

of the taste profile requires the measurement of quantitative alterations of the orange

760

sensometabolome as well as a new quality of knowledge on the interactions between

761

the tastant groups involved.

762

Dose-Response Functions of Orange Sensometabolites. In order to

763

address the non-linear relationship between concentration and bitterness intensity,

764

dose-response recordings were performed.38,39 Limonoid aglycones 1 and 2 as well

765

as the glucoside 3 followed rather different dose/response functions. Compound 1

766

reached a taste intensity of 2.8 at its maximum solubility exceeding the threshold

767

concentration by 16 times (Figure 6, A). Although nomilin (2) was water-soluble up to

768

a 32-fold threshold concentration, its maximum sensory response was evaluated with

769

an intensity of 2.2 only. Compared to 1 and 2, the taste intensity of the glucoside 3


Page 30 of 58

Page 31 of 58

770


increased only slightly with increasing DoT-factors.

771

Although polymethoxylated flavones were recently shown by cell-based

772

assays to activate hTAS2R14 as the primary bitter taste receptor with 5,6,7,8,3´,4´-

773

hexamethoxyflavone (10) showing a low EC50 value of 14 µmol/L,47 the

774

polymethoxylated flavones 4-7, 9, 10, 12, and 15 showed variations in their dose-

775

response functions strongly depending on their chemical structure (Figure 6, B). In

776

particular, the perception of 15 is reflected in a rather high slope with increased taste

777

intensities at higher concentration, e.g. 15 showed the highest taste intensity of 3.25

778

at a concentration exceeding the threshold concentration by a factor of 32. Also

779

compounds 9, 10, and 5 reached a high maximum sensory response of 3.0, 3.0, and

780

3.1, respectively, whereas compounds 4 and 6 showed comparatively flat dose-

781

response functions even at concentration exceeding the threshold by a factor of 64.

782

To investigate the bitterness enhancement of limonoids 1 and 2 by

783

polymethoxylated flavones as found by the taste re-engineering experiments (Figure

784

5), dose-response functions were recorded with solutions of 1 or 2 in the absence

785

and presence of a mixture of flavones at levels of 0.25, 0.50, and 0.75 mg/100 g,

786

respectively (Figure 6, B). Confirming the taste re-engineering data (Figure 5), the

787

bitter taste intensity of 1 was increased by the addition of polymethoxylated flavones,

788

e.g. the presence of these flavones in sub-threshold concentrations (0.75 mg/100 g)

789

increased the perceived bitterness of a solution of 1, which exceeded its bitter

790

threshold by a factor of two (DoT = 2), from 0.4 to 0.8 (Figure 6, C). Even more

791

pronounced, the bitter taste of a solution of 2 (DoT = 2) was significantly enhanced

792

from 1.0 to 1.5 in the presence of the flavones (0.5 mg/100 mL).

793

In order to investigate the influence of the flavone structure on their bitter taste

794

enhancing activity, aqueous solutions of 1 (16 µmol/L) spiked with individual

795

polymethoxylated flavones were sensorially evaluated and compared to a solution of



Page 32 of 58

796

1 in the absence of any flavone (control). While the control solution of 1 was judged

797

with an intensity of 0.9, addition of the individual flavones at a level of 0.5 mg/100 g

798

increased the perceived bitterness depending on the chemical structure (Figure 7).

799

Compounds 4, 7, and 8 were identified to affect the bitterness of 1 most intensively,

800

e.g. the presence of 7 enhanced the bitter intensity of limonin from 0.9 to 1.7. In

801

comparison, 5 did not show any significant effect in bitterness enhancement.

802

Hierarchical

Cluster

Analysis

and

Sensomics

Heatmapping

of

In order to gain a first insight into the

803

Sensometabolites in Orange Juices.

804

naturally occurring quantitative variation of the sensometabolome in orange juices,

805

limonoid aglycons (1, 2), polymethoxylated flavones (4-10, 12), limonoid glucosides

806

(3, 16, 17, 19-21), and the sugars sucrose, glucose, and fructose were quantitatively

807

determined in a total of 15 orange juices, namely OJ1-OJ15. To examine the

808

multivariate distances between the individual sensometabolites in the orange juice

809

samples, a hierarchical cluster analysis was performed on the basis of the scaled

810

DoT-factors determined for each compound. The results were visualized in a

811

sensomics heatmap that was combined with hierarchical agglomerative clustering of

812

the sensometabolites 1-10, 12, 16, 17, and 19-21. The cluster analysis quantifies the

813

degree of similarity between the sensometabolites by calculating the distance

814

between all possible pairs of molecules. The two most similar sensometabolites were

815

then grouped together and the distance measure recalculated. This iterative process

816

was continued until all sensometabolites were members of a single cluster. This

817

resulting hierarchical clustering is visually displayed as a dendrogram (Figure 8). The

818

closer the sensometabolites are to each other in the dendrogram, the smaller the

819

differences in their DoT-factor pattern throughout the set of orange juices. The

820

hierarchical analysis arranged the sensometabolites into two large clusters. Cluster 1

821

consisted of the group of polymethoxylated flavones (4-10, 12) which showed high


Page 33 of 58


822

DoT-factors in OJ5, OJ7, and OJ12 and rather low values in OJ1, OJ3, and OJ4

823

when compared to the other juice samples. The limonoid aglycons were grouped in

824

subcluster 2a with DoT-factors ranging from the lowest values of 0.5 (1) and 249

9 4 6 13 21 24 21 20 19 51 44 13

4 13 >106

n.d. n.d. 13

limonoid aglycones limonin, 1 nomilin, 2 deacetylnomilin, 22 limonoid glucosides epiisoobacunoic acid 17-O-β-D-glcp, 17 obacunon-17-O-β-D-glcp, 21 deacetylnomilinic acid 17-O-β-D-glcp, 16 limonin-17-O-β-D-glcp, 3 methyldeacetylnomilinic acid 17-O-β-D-glcp, 18 nomilin-17-O-β-D-glcp, 19 nomilinic acid 17-O-β-D-glcp, 20 1129 1130 1131 1132 1133

a

42 49 68 106 >182 >793 >702

16 15 27 17 5 404 205

Compound numbering refers to structures given in Figures 1 and 2. b Taste threshold concentrations were determined by means of a three alternative forced choice test in bottled water. c The recognition threshold concentrations were determined by means of a half-tongue test in bottled water. glcp: glucopyranoside, n.d.: not detectable.

1134 1135 1136 1137



1138 1139

Table

4.

Concentrations and Dose-over-Threshold-(DoT)-factors Compounds in Orange juices OJ1 and OJ2

taste compound (no.)a

limonin (1) nomilin (2)

13.1 (3.3) 3.8 (0.3)

group II (astringent and bitter) limonin 17-β-D-glcp (3) nomilinic acid 17-β-D-glcp (20) nomilin 17-β-D-glcp (19) deacetylnomilinic acid 17-β-D-glcp (16) epiisoobacunoic acid 17-β-D-glcp (17) obacunon 17-β-D-glcp (21) 5,6,7,8,3´,4´-hexamethoxyflavone (10) 3,5,6,7,8,3´,4´-heptamethoxyflavone (12) 5,6,7,3´,4´-pentamethoxyflavone (4) 5,6,7,8,4´-pentamethoxyflavone (6) 5,6,7,4´-tetramethoxyflavone (5) 3,5,6,7,3´,4´-hexamethoxyflavone (9) 5,7,8,3´,4´-pentamethoxyflavone (7) 3,5,7,8,3´,4´-hexamethoxyflavone (8) 5,6,7,8,3´,4´-hexamethoxyflavone-3-O-βD-glcp (15) 5,6,7,3´,4´-pentamethoxyflavone-3-O-β-Dglcp (14)

hesperidin (23) group IV (sweet) sucrose fructose glucose group IV (sour) citric acid malic acid isocitric acid

Taste

OJ2

conc. [µmol/L] (DoTbitter)

group I (bitter)

group III (velvety astringent)

of

conc. [µmol/L] (DoT)b in OJ1

1140 1141 1142 1143 1144 1145

Page 48 of 58

2.1 (0.5) 0.1 (

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