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Aroma Stability of Lemon-Flavored Hard Iced Tea Assessed by Chirality and Aroma Extract Dilution Analysis Fang Yuan, Fei He, Yanping L. Qian, Jia Zheng, and Michael C. Qian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01776 • Publication Date (Web): 18 Jun 2016 Downloaded from http://pubs.acs.org on June 22, 2016

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Journal of Agricultural and Food Chemistry

Aroma Stability of Lemon-Flavored Hard Iced Tea Assessed by Chirality and Aroma Extract Dilution Analysis

Fang Yuan, Fei He, Yanping Qian, Jia Zheng and Michael C. Qian*

Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331

*To whom correspondence should be addressed

M.C.Q, Phone: (541) 737-9114. Fax: (541) 737-1877. E-mail: [email protected].

ACS Paragon Plus Environment


1

Abstract

2

The aroma of fresh and aged lemon-flavored hard tea was investigated by aroma extract

3

dilution analysis (AEDA), quantitative comparison, and two-dimensional chirality

4

analysis. Aroma extract dilution analysis of fresh hard tea samples showed 3-

5

methylbutanal, isoamyl alcohol, β-damascenone, β-ionone, 2-phenylethanol, 4-hydroxy-

6

2,5-dimethyl-3(2H)-furanone and vanillin could be the most important aroma

7

contributors to the hard tea due to their high FD value. The analysis of the aged hard tea

8

samples did not reveal new compound formation during storage, however, compared with

9

fresh samples, the flavor dilution value changed substantially in the aged samples. Both

10

AEDA and quantitative analysis demonstrated that β-damascenone increased

11

substantially in aged samples, whereas terpene aldehydes decreased substantially after

12

storage. In addition, the FD value of linalool decreased dramatically in aged samples.

13

Two-dimensional GC-MS chirality analysis revealed the FD value decrease of linalool in

14

aged samples was largely due to the transformation of (R)-linalool to (S)-linalool, which

15

has a higher sensory threshold.

16

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Key words: tea beverage; AEDA; chirality; linalool; two-dimensional GC-MS


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Introduction

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Flavored alcoholic beverages are those that between the alcohols and soft drinks. They

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were first introduced into the alcohol market of US in the early 1980s and were often

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called “low alcohol coolers”, “flavored alcoholic beverages”, “flavored malt beverages”,

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“alco-pops”, “malternatives”, and “alcohol refreshers”.1 These beverages typically have

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an alcohol content of about 5%. Hard ice tea is a ready-to-drink tea beverage containing

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alcohol. The product is typically flavored with artificial or natural flavorings. The product

25

is typically formulated with tea extract, and lemon flavoring is commonly used in the

26

formulation. Due to the low pH and complexity of the beverage, flavor stability is a great

27

concern for both manufacturer and consumer.

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Volatile components play an essential role in the aroma characteristics and flavors of tea.

29

To date about 600 constituents have been characterized in tea leaves or in the tea

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beverage.2 Aroma extract dilution analysis (AEDA) has been used to identify the odor-

31

active compounds in tea since 1990s.3 Masuda and Kumazawa4 reported that β-

32

damascenone and linalool were the most odor-active compounds in the tea beverage. In

33

addition, geraniol, dimethyl trisulfide, 2-methoxy-4-vinylphenol, methyl salicylate,

34

phenylacetaldehyde, (E,Z)-2,6-nonadienal, methional, and 3-methylbutanal were also key

35

contributors. Schuh and Schieberle2 reported that vanillin, 4-hydroxy-2,5-dimethyl-

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3(2H)-furanone, 2-phenylethanol, and (E,E,Z)-2,4,6-nonatrienal had the highest flavor

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dilution (FD) factors among 24 odor-active compounds detected in Darjeeling Black Tea.

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Hai-Peng Lv et al.5 reported that in Pu-erh tea, 1,2-dimethoxybenzene, 1,2,3-



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trimethoxybenzene, 1,2,3-trimethoxy-5-methylbenzene, 4-ethyl-1,2-dimethoxy-benzene,

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β-ionone, β-linalool, linalool oxides, decanal were responsible for the special flavor.

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Among flavored hard iced tea beverages, lemon flavor is the most popularly used in the

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industry. It is well documented that terpene hydrocarbons (i.e. pinenes, limonene, and

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terpinenes) and oxygen-containing terpenoids (i.e. citronellal, linalool, citral, neral and

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geranial) are the main contributors to the odor of lemon flavor.6 Among them, citral (3,7-

45

dimethyl-2,6-octadienal) is one of the most important flavor compounds in citrus oils,

46

and is widely used in foods and beverages,6 while other volatile components had less

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impact but providing the complexity to the lemon flavor. It is well-known that lemon

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juice, lemon natural extract or lemon oil are unstable, they deteriorate quickly via

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autoxidation in the presence of heat, acids and UV radiation.7, 8 Flavor stability is a major

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concern when fruit juices or fruit flavors are employed in ready-to-drink tea beverages

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because of the low pH and other active ingredients present in the beverage.

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It has long been recognized that chiral enantiomers of aromatic compounds may have

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different aroma character or aroma intensity due to their varied interaction with olfactory

54

chemoreceptors.9 For example, the odor threshold of the (R)-linalool (perceived as

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woody and lavender-like) is about 80 times lower than that of the (S)-enantiomer

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(perceived as sweet, ﬂoral, petitgrain-like).10 Friedman and Miller11 reported that the

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characteristic odor for (S)-(+)-carvone is caraway with an odor threshold of 130 µg/L in

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water, whereas (R)-(-)-carvone is spearmint-like with an odor threshold of 2 µg/L. (R)-

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(+)-limonene has an odor of orange but (S)-(-)-limonene smells like lemon.11 Depending

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on the pH, temperature, state (i.e., liquid or solid), and other factors, many chiral


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molecules can transform from one form to another, altering the aroma quality of the

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products.

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Two-dimensional GC-MS with “heart-cut’ technique has been widely used for

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complicated plant volatile and food extract analysis. This technique is particularly useful

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for enantiomer analysis of volatile aroma compounds. In this application, the volatiles

66

are separated in the first analytical column, and only those components whose

67

enantiomers need to be determined are “heart-cut” and transferred to the second GC

68

column to resolve the chiral enantiomers. Since only the interested compounds are

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transferred to the second chiral column, the technique greatly reduces peak overlap and

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interference, allowing correct analysis of the chiral enantiomers in complicated sample

71

matrices.9, 12

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The aim of this study is to understand the aroma change of lemon-flavored hard iced tea.

73

The aromas of the fresh and aged samples were evaluated by AEDA and quantitative

74

analysis. Racemization of odor-active chiral compounds was also determined before and

75

after storage. The result of this study can provide a foundation for improvements in the

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flavor and shelf life stability of lemon-flavored hard iced tea beverages.

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Materials and Methods

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Reagents and Chemical Standards

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Dichloromethane was HPLC grade (EMD, Gibbstown, NJ) and was redistilled before

80

use. Water was obtained from a Milli-Q purification system (Millipore, North Ryde,

81

NSW, Australia).



82

Standards of volatile compounds were purchased from commercial sources. 3-

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Methylbutanal (97%), 2-methylbutanal (≥97%), diacetyl (2,3-butanedione, 97%), ethyl

84

butanoate (>98%), hexanal (98%), eucalyptol (99%), isoamyl alcohol (>98%), vanillin

85

(98.0%), nerol (98.0%), geraniol (98.0%), p-cymene (99%), 3-hydroxybutanone (95%),

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octanal (99%), furfural (99%), (Z)-3-hexenol (98%), acetic acid (99.5%), methional

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(98%), furfural (99%), 3-isobutyl-2-methoxypyrazine (99%), butanoic acid (99%), 3-

88

methylbutanoic acid (99%), nerol (98%), benzeneacetaldehyde (≥99%), citral (90%, neral

89

and geranial mixture), β-citronellol (95.0%), β-damascenone (≥90.0%), 2-phenylethanol

90

(99.0%), β-ionone (90.0%), phenol (≥96%), p-cresol (99%), eugenol (98%), 4-

91

methylguaiacol (99%) and 4-ethylphenol (98%) were purchased from Sigma-Aldrich (St.

92

Louis, MO). 4-Vinylguaiacol (97.0% with 0.01% BHT) and 4-vinylphenol (10% solution

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in propylene glycol) were purchased from Alfa Aesar (ward Hill, MA). 4-Hydroxy-2,5-

94

dimethyl-3-furanone (99%), maltol (99%), 3-ethylphenol (95%) and geranyl acetate

95

(90%) were purchased from TCI America (Cambridge, MA). Enantiomer standards

96

including S-(-)-limonene (96%), R-(+)-limonene (97%), α-terpineol (90.0%, mixture of

97

enantiomers), R-(-)-linalool (≥95%) and (+)-terpinen-4-ol (≥98.5%, enantiomeric ratio:

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~2:1) were purchased from Sigma-Aldrich (St. Louis, MO).

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Aroma Extraction of Hard Iced Tea Sample

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Two freshly manufactured lemon-flavored iced tea samples (pH 3.2, with 5% alcohol by

101

volume) were obtained commercially. Two other samples from the same manufacturer

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were aged for one year under refrigerated conditions. Each sample (710 mL, 24 oz) was

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mixed with 75 mL of freshly distilled dichloromethane in a separatory funnel, and shaken


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vigorously for 30 min. The lower layer was collected, and the aqueous layer was

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extracted again with another 75 mL of dichloromethane. The resulted solvents were

106

pooled, and the volatiles were isolated using a solvent-assisted flavor evaporation (SAFE)

107

unit described by Engel et al. 13 The volatiles were evaporated with dichloromethane at 50

108

°C under vacuum, and was condensed in round bottom flask merged in liquid nitrogen.

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The distillates were dried over anhydrous sodium sulfate and concentrated to 0.5 mL

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under a stream of nitrogen.

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Compound Identification by GC-Olfactometry

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GC-Olfactometry analysis was used to identify the potential odor-active compounds in

113

the aroma extract from the hard iced tea samples. GC-O analysis was performed on an

114

Agilent 6890 gas chromatograph equipped with an Agilent 5973 mass-selective detector

115

(MSD) and a sniffing port (ODP 2, Gerstel, Germany). A DB-WAX capillary column (30

116

m × 0.32 mm i.d.; 0.25 µm film thickness, Agilent, Santa Clara, CA) was used for

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chromatographic separation. Helium was used as carrier gas at a constant flow rate of 2

118

mL/min. The column effluent was split 1:1 into the MSD and the sniffing port via two

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deactivated fused silica capillaries (0.1 mm i.d.). Sample (1 µL) was injected into the GC

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injector in splitless mode. The GC injector temperature was 230 °C. The oven

121

temperature was programmed at 40 °C and held for 2 min, and then increased to 230 °C

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at a rate of 4 °C/min, with a 10-min hold at the final temperature. The MS transfer line

123

and ion source temperature were 280 °C and 230 °C, respectively. The mass selective

124

detector in full scan mode was used for acquiring the data. Electron ionization mass



125

spectrometric data from m/z 35 to 300 were collected, with an ionization voltage of 70

126

eV.

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The identification method for each compound was showed in Table 1. Most of the

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volatile compounds were identified by comparing their mass spectra with those in the

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Wiley 275.L Database (Agilent Technologies Inc.) and retention indices (RIs) with those

130

of authentic standards available in the laboratory using the same instrument. When

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authentic standard was not available, the compound was tentatively identified by

132

comparing their aroma and RI with the value reported in literature using same GC

133

column. RIs were calculated after analyzing C6-C20 n-alkane series (Supelco, Bellefonte,

134

PA) under the same chromatographic conditions.

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Aroma Extract Dilution Analysis (AEDA)

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After the potential odor-active compounds were identified by GC-O analysis, AEDA was

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used to evaluate the aroma potency of these compounds. Aroma extracts of the hard iced

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tea samples were stepwise diluted with dichloromethane at 1:1 ratio. The AEDA was

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performed by three experienced panelists and duplicate analyses were performed by each

140

panelist. Each dilution was submitted to GC-O analysis on the DB-WAX column. The

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flavor dilution (FD) factor of each compound was determined as the maximum dilution at

142

which the odorant could be perceived.

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Quantitation of Volatile Compounds by Stir Bar Sorptive Extraction (SBSE)-GC-MS

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For quantification, the standard solutions of all pure chemical compounds were prepared

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by diluting the stock solution of known amount of authentic standards in synthetic matrix


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(0.1 M citrate buffer, pH 3.2, containing 5% ethanol, v/v) to give a range of

147

concentrations. The standard solutions were analyzed using the same procedure as

148

described for hard iced tea samples. The calibration curve for each target compound was

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built up by plotting the selected mass ion abundance ratio of target compound with

150

internal standard against the concentration ratio. Standard calibration curves were

151

obtained through ChemStation Software (Agilent Technologies, Santa Clara, CA) and

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were used to calculate the concentrations of volatile compounds in hard iced tea samples.

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Duplicate analysis was performed for each sample.

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A SBSE-GC-MS method with ethylene glycol-silicone (EG) coated stir bar (0.5 mm film

155

thickness, 10 mm length, Gerstel Inc., Baltimore, MD) was employed for the quantitation

156

of volatile compounds in the standard as well as hard iced tea samples.14 Ten mL of each

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sample was mixed with 10 mL of saturated NaCl solution in a 40 mL vial. An aliquot of

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20 µL internal standard (4-octanol, 122 mg/L) and the EG stir bar were added. The vial

159

was tightly capped and stirred (1000 rpm) for 3 h at room temperature. After extraction,

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the EG stir bar was removed from the sample, rinsed with Milli-Q water, dried with

161

Kimtech wipers (Kimberly-Clark Professional Inc., Roswell, GA), and transferred into a

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thermal desorption tube for GC-MS analysis.

163

Analysis of volatile compounds were performed on an Agilent 7890 gas chromatograph

164

coupled with a 5975 mass selective detector, a Gerstel MPS-2 multipurpose autosampler

165

with a Gerstel Thermal Desorption Unit (TDU), and a CIS-4 cooling injection system

166

(Gerstel Inc.). The analytes were thermally desorbed at the TDU in splitless mode,

167

ramping from 20 °C to 230 °C at a rate of 120 °C/min, and held at the final temperature



168

for 2 min. The CIS-4 was cooled to -80 ºC with liquid nitrogen during the sample

169

desorption, and then heated at 10 °C/s to 250 °C for 10 min. Solvent vent mode was used

170

during the injection with a split vent flow of 50 mL/min. A ZB-WAX capillary column

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(60 m × 0.25 mm i.d., 0.5 µm film thickness, Phenomenex, Torrance, CA) was used. The

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oven temperature program was set at 40 ºC for 2 min, raised to 230 ºC at 4 ºC/min, hold

173

for 20 min. A constant helium flow of 2 mL/min was used. The MS transfer line and ion

174

source temperature were 280 °C and 230 °C, respectively. The mass selective detector in

175

the full scan mode was used for acquiring the data. Electron ionization mass

176

spectrometric data from m/z 35 to 300 were collected, with an ionization voltage of 70

177

eV.

178

Chiral Analysis by Two Dimensional GC-MS

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Two dimensional GC-MS analysis was performed on a Mach system with LTM columns

180

(Gestel, USA). Two mL of hard iced tea sample was diluted with 8 mL of citric acid

181

buffer (0.1 M, pH 2.5), and 20 µL of internal standard (4-octanol, 34 mg/L) was added.

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The extraction and injection was conducted by an autosampler (Gerstel, USA). Samples

183

in the vials were incubated at 50 °C for 5 min, with an agitation speed of 250 rpm. The

184

volatile compounds were extracted for 50 min using a 50/30 µm DVB/CAR/PDMS fiber

185

(2 cm, Supelco, Bellefonte, PA). Volatile compounds were first separated on a DB-WAX

186

column (30m × 0.25mm i.d., 0.5 µm film thickness, Agilent Technologies), then the

187

targeted compounds were “heart-cut” and transferred to a second column coated with

188

30% hepatkis (2,3-di-O-methyl-6-O-t-butyl dimethylsilyl)-β-cyclodextrin (Cyclosil B, 30

189

m x 0.25 mm i.d., 0.25 µm film thickness, Agilent Technologies) for further separation.


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The first column oven temperature was held isothermally at 80 °C for 2 min and then

191

increased to 230 °C at a rate of 4 °C/min, with a 20-min hold at the final temperature.

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The second column oven temperature was programmed at 80 °C for 20 min and then

193

increased to 150 °C at a rate of 2 °C/min, with a 3-min hold at the final temperature. The

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retention time of limonene, linalool, terpinen-4-ol and α-terpineol on wax column was

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9.95 min, 20.25 min, 23.8 min and 26.5 min respectively, therefore the cutting window

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was set to 9.5-10.5 min, 19-22 min, 23-24.5 min and 26-30 min. In addition, a cutting

197

interval of 15-16 min was chosen to cut internal standard. The isomeric ratio was

198

determined using the relative total mass ion abundance of the compound. Each sample

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analysis was performed in triplicates.

200

The R- and S-enantiomers of limonene, linalool and terpinen-4-ol were identified by

201

comparing retention time with authentic standards. α-Terpineol enantiomers were

202

identified by comparing with published elution order15 from the same column.

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Results and Discussion

204

Odor-active compounds in hard iced tea

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Total 49 odor-active compounds were detected in the AEDA. They mainly belong to the

206

categories of alcohols, esters, aldehydes, furanones, volatile phenols, C13-norisoprenoids,

207

and terpenoids. Comparing the two fresh hard iced tea samples, the major odorants were

208

similar (Table 1). According to the FD value, the most important aroma compounds in

209

the fresh hard iced tea were isoamyl alcohol (FD>64), linalool (FD=64), 2-phenylethanol

210

(FD=64), followed by 3-methylbutanal (FD=32), nerol (FD=32 in sample 1 and FD=16



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in sample 2), geraniol (FD=32), β-ionone (FD=32), 4-hydroxy-2,5-dimethyl-3(2H)-

212

furanone (FD=32), 4-vinylphenol (FD=16 in sample 1 and FD=32 in sample 2) and

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vanillin (FD=32). Overall, the FD value of the major aroma compounds between sample

214

1 and sample 2 were very close, considering the variation involved in AEDA analysis.

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Compared with the fresh samples, the aged samples did not contain any extra odor-active

216

compounds (or off-flavor compounds). The main difference between fresh and aged

217

samples was the change of FD values of the odor-active compounds (Table 1).

218

Considering the possible variations in the AEDA study, to fully understand the flavor

219

change, the major aroma-active compounds were further quantitated and their odor

220

activity values (OAVs) were also determined (Table 2). Generally, odor active

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compounds with high OAVs are more likely to be major contributors to hard iced tea

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aroma, although aroma synergy and suppression probably exist. Compounds with high

223

OAV (>20) in the fresh hard iced tea samples were p-cymene, nerol, β-damascenone, β-

224

ionone and 4-vinylphenol. But in the aged samples, only the OAVs of β-damascenone, β-

225

ionone and 4-vinylphenol were still above 20. Both the AEDA analysis and quantitative

226

results demonstrated the aroma profile change, and the change was compound dependent

227

as discussed below.

228

Alcohols

229

Isoamyl alcohol, (Z)-3-hexenol and 2-phenylethanol were three odor-active alcohols in

230

the hard iced tea samples. Among them, 2-phenylethanol and isoamyl alcohol had high

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FD values in both fresh and aged hard iced tea samples. (Z)-3-hexenol originated from

232

the tea ingredient. It contributed to the grassy odor of the beverage, but its FD value was


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not high (FD=4). Isoamyl alcohol and 2-phenylethanol were mainly derived from

234

alcoholic fermentation thus had very high concentration in the samples.

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Comparing the fresh samples with aged samples, the concentrations of (Z)-3-hexenol and

236

2-phenylethanol did not change according to both AEDA and quantitation data,

237

indicating a good stability of these two compounds during storage. There was a 37~41%

238

reduction of isoamyl alcohol concentration in the aged samples compared to the fresh

239

ones. The decreasing isoamyl alcohol was unexpected in this study. It could be partially

240

resulted from esterification during storage since a very small increase of isoamyl acetate

241

was observed in aged samples (data not shown) but was not equivalent to the amount that

242

isoamyl alcohol decreased. Decrease of isoamyl alcohol in the aged samples might be

243

also due to variability of production because isoamyl alcohol is one of the major

244

fermentation byproduct from the alcohol base.

245

Esters

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Ethyl butanoate was the only odor-active ethyl ester found in hard iced tea sample. It was

247

generated during fermentation and contributed to the fruity aroma in the samples. The

248

concentration of ethyl butanoate decreased 20~51% in aged samples compared to fresh

249

samples, which was similar with wine aging that ethyl esters decreased due to the

250

hydrolysis at low pH.16 Geranyl acetate and geranyl formate were two terpene esters

251

identified in hard iced tea, contributing to the fruity and floral type of odor. Geranyl

252

acetate concentration were 95~99% lower in aged sample than fresh one. Due to the low

253

concentration, geranyl formate were not quantitated in this study, but according to the FD

254

value in the AEDA, it was also showed decreased FD value in the aged samples. Geranyl



255

esters could go through acid-catalyzed reactions, resulting in a variety of degradation

256

products including linalool, α-terpineol and geraniol.17

257

Aldehydes

258

Several aldehydes, such as phenylacetaldehyde, 4-heptanal, 2-methylbutanal, 3-

259

methylbutanal, 1-hexanal, octanal, methional and furfural, were found in the hard iced tea

260

samples. 1-Hexanal contributed to the typical green or grassy odor of the tea.2

261

Phenylacetaldehyde, methional and 3-methylbutanal has also been suggested as key

262

contributors to the aroma of the tea drink.2 However, in our study, most of them did not

263

show high FD values in the AEDA except for 2-methylbutanal and 3-methylbutanal,

264

which contributed to a malty odor. Among them, hexanal, octanal, furfural and

265

phenylacetaldehyde were quantitated and the result showed a large decrease, which

266

possibly due to oxidation during extended storage.

267

Furanones

268

4-Hydroxy-2,5-dimethyl-3(2H)-furanone, 4,5-dimethyl-3-hydroxy-2(5H)-furanone

269

(sotolon) and 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) have cotton candy, caramel-

270

like aroma. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone in hard iced tea showed very high

271

FD value (FD=32) due to its low sensory threshold (5 µg/L).18 Our result was in

272

agreement with previous report that the 4-hydroxy-2,5-dimethyl-3(2H)-furanone was one

273

of the important odor-active compound with high FD value found in Darjeeling Black

274

Tea.2 According to the FD value, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and maltol

275

was not different in fresh and aged samples. Sotolon and maltol had lower FD value


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compared to 4-Hydroxy-2,5-dimethyl-3(2H)-furanone. These three compounds could not

277

be reliably quantified by SBSE-GC-MS method employed, possibly due to the thermal

278

instability of these molecules.

279

Volatile phenols

280

There were several volatile phenols among the identified odorants, among which 4-

281

vinylphenol had highest FD factors (FD=16~32). 4-Vinylguaiacol has a clove like aroma.

282

In wine research, the decrease of 4-vinylguaiacol and 4-vinylphenol has been extensively

283

studied and has been partly attributed to the slow acid-catalyzed addition of ethanol,

284

yielding 4-(1-ethoxyethyl)-phenol and 4-(1-ethoxyethyl)-guaiacol, respectively.19

285

However, in hard iced tea samples, the concentration of 4-vinlyphenol did not change

286

during the experimental period of time, indicating these compounds were relatively stable

287

in the samples. Other volatile phenols, such as p-cresol, 4-methylguaiacol, 3-ethylphenol,

288

and 4-ethylphenol showed less intense FD values, although the similarities of their odors

289

may make them important odor contributors.

290

C13-norisprenoids

291

Two C13-norisoprenoids, β-damascenone and β-ionone, were detected in the AEDA

292

study. Both of them were important characteristic flavor compounds in tea infusions.20 β-

293

Ionone concentration was slightly lower in the aged samples but did not result in

294

difference in AEDA. It was interesting that β-damascenone concentration increased

295

dramatically in the aged samples. Recently, three major glycosidic precursors of

296

damascenone, 9-O-β-D-glucopyranosyl-megastigma-6,7-dien-3,5,9-triol, 9-O-β-D-



297

glucopyranosyl-3- hydroxy-7,8-didehydro-β-ionol, and 3-O-β-D-glucopyranosyl-3-

298

hydroxy-7,8-didehydro-β-ionol were isolated and identified in green tea infusions.21 It

299

has been reported that β-damascenone could be generated in canned and bottled green

300

and black teas during the heat processing (pH 5.4 and 120 °C for 10 min),22 suggesting

301

the hydrolysis or transformation of the glycoconjugate precursors at high temperatures.

302

Our result indicated that the hydrolysis of precursors could also happen at lower pH (pH

303

3.2) during storage, without heating. It demonstrates that damascenone is liberated as an

304

off-flavor not only during manufacturing of bottled and canned green tea beverages,23 but

305

also during storage, as a consequence of the hydrolysis of the glycoconjugate precursors.

306

Terpenoids

307

Terpenoids were the group of compounds mainly responsible for the lemon, floral, and

308

minty aroma in the hard iced tea samples. Many of them showed significant lower FD

309

value in aged sample compared to the fresh in the AEDA, which is in agreement with

310

quantitation data (Table 2). Citronellol, nerol, geraniol, neral, geranial are described as

311

fruity, lemon-like and citrus. The decreasing of terpene alcohols, such citronellol, nerol

312

and geraniol, varied from 12~45%, while terpene aldehydes, such as neral and geranial,

313

decreased 99~100% compare to the fresh samples. Our result was in agreement with

314

previous study that citral (neral and geranial) decomposes rapidly during storage at acidic

315

pH by a series of cyclisation and oxidation reactions.24 Eucalyptol and p-cymene were

316

two terpene compounds detected in AEDA that had mint and eucalyptus-like aroma.

317

Eucalyptol slightly increased in the aged sample (3.5~11%), indicating it might be a

318

degradation product that responsible for the undesirable off-odors. Although it was


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319

reported that p-cymene was one of the degradation products from citral,25 the

320

concentration of p-cymene decreased 88~95% compared to fresh samples. Another citral

321

degradation product, p-cymen-8-ol,25 was observed only in the aged samples (data not

322

shown) but was not odor-active. It was remarkable that α-terpineol and terpinen-4-ol

323

concentration increased in both aged samples compared to fresh one, which was in

324

agreement that α-terpineol and terpinen-4-ol were degradation product of d-limonene and

325

linalool in some fruit and fruit juices,26-28 as well as hydrolysis of terpene alcohol

326

glycosides. α-Terpineol could impart a stale, musty or piney odor to the product,29 which

327

might be an off-flavor in hard iced tea in extended storage. Although increased in

328

concentration, terpinen-4-ol was not detected by AEDA in all samples, therefore was not

329

considered as odor-active compound.

330

For linalool, the concentration difference between fresh and aged sample was very small

331

(Table 2) but the FD value of linalool changed significantly (Table 1). To test the

332

hypothesis of possible racemization of linalool during storage, the enantiomeric

333

composition of the chiral compounds in the hard iced tea samples was further analyzed

334

by 2D-GC-MS.

335

Enantiomeric composition

336

The enantiomeric composition of limonene, linalool, terpinen-4-ol and α-terpineol were

337

determined in both fresh and aged hard iced tea samples by 2D-GC-MS, and their OAV

338

(odor activity value) was calculated based on their odor thresholds in the literature (Table

339

3). D-limonene or the (R)-(+)-isomer was present in greatest abundance (91~96%). This

340

is to be expected because it is well known that natural products such as citrus oil contain



341

majorly d-limonene. d-Limonene gives fresh citrus-like odor characteristics in the

342

essential oils, while l-limonene has a harsh, turpentine note.30 However, due to the high

343

odor threshold of d-(+)-limonene (1.20 mg/L in water),10 its contribution to the lemon

344

odor of the hard iced tea was very limited as shown by AEDA study (FD=1), although its

345

OAV was higher than 1 in the fresh samples. Linalool is one of the major flavor impact

346

compounds in the hard iced tea samples. Linalool in natural flavor is usually present as a

347

mixture of the (R)-(−)-isomer and (S)-(+)-isomer in different ratios. Although the total

348

amount of linalool in hard iced tea samples barely changed after storage, the enantiomeric

349

ratio was different for fresh and aged samples. In fresh samples, there was more (R)-(−)-

350

linalool than the (S)-(+)-isomer, suggesting conversion of (R)-(−)-linalool to (S)-(+)-

351

linalool during storage. The odor threshold of the former enantiomer is about 9 times

352

lower than the latter one (0.8 and 7.4 µg/L, respectively10). Thus, at similar

353

concentrations, (R)-(−)-linalool should make a greater aroma contribution than (S)-(+)-

354

linalool. Comparing the OAV, the conversion of (R)-(-)-isomer resulted in greater OAV

355

decrease compared to the increase of (S)-(+)-isomer. Racemization of (R)-(-)-linalool

356

during storage should consequently result in a reduction of the floral and citrus aroma of

357

the tea. The enantiomeric compositions of limonene, terpinen-4-ol and α-terpineol were

358

very similar in fresh and aged samples.

359

In summary, our results indicated the aroma change of lemon-flavored hard iced tea was

360

due to the large decrement of terpene compounds and racemization of (R)-linalool, which

361

led to a significantly lower aroma intensity of lemon, minty, and fresh flavor in the hard

362

iced tea product, and the increase of β-damascenone. Alcohols and volatile phenols were

363

relatively stable in the iced tea product during storage.


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364

Abbreviations Used

365

SPME, solid-phase microextraction; SBSE, stir bar sorptive extraction; EG，ethylene

366

glycol； TDU， thermal desorption unit; GC-MS, gas chromatography-mass

367

spectrometry; GC-FID, gas chromatography with flame ionization detection; GC-O, gas

368

chromatography-olfactometry; GC-MS-O, gas chromatography- mass spectrometry-

369

olfactometry; AEDA, aroma extract dilution analysis; FD, flavor dilution; RI, retention

370

index; OAV, odor activity value.



371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

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Table 1 Potent odorants in hard iced tea detected by AEDA on DB-WAX column RIZB-Wax 887 902 910 1028 1053 1181 1190 1192 1202 1238 1281 1320 1394 1431 1465 1487 1532 1547 1576 1642 1651 1655 1672 1705 1714 1718 1743 1746 1833 1849 1900 1950 1960 1966 1973 2024 2067 2080 2109 2151 2169 2172 2146 2194 2204 2230 2279 2427 2583

Compound 3-Methylbutanal 2-Methybutanal Diacetyl Ethyl butanoate Hexanal Eucalyptol Limonene Isoamyl alcohol p-Cymene 4-Heptenal 3-Hydroxybutanone Octanal (Z)-3-Hexenol Acetic acid Methional Furfural 3-Isobutyl-2methoxypyrazine Linalool 2,6-Nonadienal Butanoic acid Phenylacetaldehyde 3-Methylbutanoic acid Neral α-Terpineol Geranyl acetate Geranial Citronellol Nerol β-Damascenone Geraniol 2-Phenylethanol β-Ionone Unknown p-Methylguaiacol Geranyl formate 4-Hydroxy-2,5-dimethyl3-furanone (Z)-2-Hexenoic acid p-Cresol Eugenol Maltol 4-Ethylphenol 3-Ethylphenol Sotolon 4-Vinylguaiacol Unknown Unknown Unknown 4-Vinylphenol Vanillin

Sample 1 FD factor Fresh Aged 32 32 16 16 8 16 8 4 4 2 16 16 1 1 >64 64 4 2 2

Aroma Stability of Lemon-Flavored Hard Iced Tea ... - ACS Publications

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