Quantitation and Seasonal Variation of Key Odorants in Propolis

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Chemistry and Biology of Aroma and Taste

Quantitation and Seasonal Variation of Key Odorants in Propolis Monika Tomaszewski, Melissa Dein, Ari Novy, Thomas G Hartman, Martin Steinhaus, Curtis R Luckett, and John P Munafo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05965 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Journal of Agricultural and Food Chemistry

Quantitation and Seasonal Variation of Key Odorants in Propolis

Monika Tomaszewski,1 Melissa Dein,2 Ari Novy,3 Thomas G. Hartman,1 Martin Steinhaus,4 Curtis R. Luckett,2 and John P. Munafo Jr.*,2

1 Department

of Food Science, Rutgers-The State University of New Jersey, 65 Dudley Rd, New

Brunswick, New Jersey 08901, United States 2

Department of Food Science, University of Tennessee, Knoxville, Tennessee 37996, United States

3 San

Diego Botanic Garden, 230 Quail Gardens Dr., Encinitas, CA 92024; Department of

Anthropology, University of California-San Diego, La Jolla, CA 92093; Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20002, USA 4 Leibniz-Institute

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

Meitner-Str. 34, 85354 Freising, Germany

* Corresponding author (J.M.): Phone: 865-974-7247. Fax: 865-974-7332. E-mail: [email protected]

1 ACS Paragon Plus Environment


1

ABSTRACT: Propolis is a fragrant material produced by bees and is commonly used as an

2

ingredient in the food, beverage, and consumer goods industries. Application of a comparative

3

aroma extract dilution analysis (cAEDA) to volatiles isolated from propolis of three consecutive

4

years afforded 48 odorants with flavor dilution (FD) factors ≥ 4, including 21 compounds not

5

previously reported in propolis. Despite differences in FD factors of some compounds, the overall

6

temporal variation in the odorants was low. Compounds with FD ≥ 64 were quantitated by stable

7

isotope dilution assays (SIDAs) and odor activity values (OAVs) were calculated. Twenty-two

8

compounds showed OAVs ≥ 1, including (E)-isoeugenol (clove; OAV 3700), linalool (floral;

9

OAV 380), butanoic acid (sweaty, rancid; OAV 370), and 3-phenylpropanoic acid (floral; OAV

10

270). An odor reconstitution model prepared from deodorized beeswax and the 22 odorants in their

11

natural concentrations closely matched the olfactory profile of authentic propolis. The results of

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this study will help to establish a basis for future research on the variability of propolis sourced

13

from different geographical locations, produced by different bee species, and collected from

14

different botanical sources, all of which is largely unknown.

15

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KEYWORDS: Propolis, solvent-assisted flavor evaporation, gas chromatography-

17

olfactometery, aroma extract dilution analysis and stable isotope dilution assay.


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INTRODUCTION

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Propolis is a fragrant, sticky, and resinous plant-derived material collected by bees as a

20

caulking, sealing, lining, strengthening, and preserving material for hive construction.1 Propolis is

21

found inside the hive and around its entrance, and may have a repelling or masking effect that

22

protects bee colonies from certain pests and diseases. Because honeybee populations are confined,

23

and the bees live in close proximity to one another, illness can easily spread from one bee to the

24

entire hive. It is postulated that good hive health may be maintained, in part, by the antimicrobial

25

properties of propolis, resulting in the reduction of microbial growth on hive walls.2 Propolis is

26

collected by all species of Apis, as well as by stingless bees such as Melipona and Trigona species.3

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Foraging bees collect propolis substrates from the resinous exudates of woody trees and shrubs. It

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is thought that exudates of the deciduous tree genus Populus are preferred by bees, however bees

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must collect exudates from a variety of plant species, depending on geography and seasonal

30

availability.4 For example, there is evidence of propolis collection from Pinus spp. and desert

31

composites as well.5, 6

32

Propolis has a long history of therapeutic use by both Old and New World civilizations. In

33

ancient Greece and Rome, its use as an antiseptic and cicatrizant was noted by prominent

34

philosophers and physicians, including Aristotle, Dioscorides, and Pliny.7 In ancient Egypt,

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propolis was used for embalming preparations, while the Inca of the Americas used propolis as an

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antipyretic.8 In recent years, preparations made from propolis have become increasingly popular

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for functional food, dietary supplement, and cosmetic applications. Propolis is commonly taken

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internally in the form of capsules, throat sprays, and tinctures, and can also be topically applied to

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the skin in the form of lotions and ointments.8 Today, the antimicrobial properties of propolis are



40

well documented.9 The substance is also being investigated for anti-cancer properties, immune

41

activation, and other clinical uses.10, 11

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Although propolis does not have a uniform composition, some studies have indicated

43

remarkable similarity between propolis of different origins, however some propolis, such as

44

Brazilian propolis, appear to have unique qualities including anticancer and immunomodulatory

45

properties.5, 12, 13 Propolis is commonly brown in color but is also found in shades of grey, yellow,

46

green, red, and black, depending on its age and botanical source. The chemical composition of

47

propolis varies from sample to sample in regard to both volatile and non-volatile components.

48

Over 180 compounds have been identified as constituents of propolis,8 of which several have been

49

identified as biologically active, including flavonoids and phenolics. Some additional chemical

50

constituents previously identified include cinnamyl alcohol, cinnamic acid, vanillin, benzyl

51

alcohol, benzoic acid, caffeic acids, ferulic acids, phenolic triglycerides, pterostilbene, eugenol,

52

and caffeic acid pentenyl esters.3

53

In addition to the significant body of scientific research conducted on the biological

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activities of non-volatile components present in propolis, fewer investigations have been aimed

55

at characterizing the volatiles.14-23 Gas chromatography-mass spectrometry (GC-MS) analyses

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have shown that a single propolis sample may contain over 150 volatiles.22 In addition to its

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purported health-promoting properties, one important factor that contributes to the popularity of

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propolis is its pleasant odor. Propolis has a highly fragrant scent that can be described as similar

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to beeswax and honey with complex spicy, herbal, and floral nuances. Although some studies

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have focused on volatiles, limited work has been conducted on odor-active compounds. In 2010,

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Yang and co-workers employed gas chromatography-olfactometry (GC-O) to identify 44 odor-

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active compounds in propolis collected from different regions of China.14 However, at the


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present there is little understanding of the influence of environmental factors on propolis quality

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and stability from a single source. We are not aware of any prior research designed to observe

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the temporal variability of propolis in a single beehive over consecutive years, nor quantitation

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of key odorants present in propolis.

67

Due to the increasing popularity of propolis as an ingredient in the food, beverage, and

68

consumer goods industries, a better understating of the key odorants responsible for its pleasant

69

aroma are needed to lay the groundwork for future studies aimed at ingredient standardization

70

and quality control. Therefore, the aim of this investigation was: 1) identify the key odorants

71

present in propolis and gain insight into the variability between years by performing a

72

comparative aroma extract dilution analysis (cAEDA) on the volatile isolates generated via

73

solvent-assisted flavor evaporation (SAFE); 2) quantitate the odorants with high FD factors by

74

stable isotope dilution assays (SIDAs) and calculate odor activity values (OAVs); and 3)

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duplicate the odor of propolis using the quantitative results in combination with sensory

76

experiments.

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MATERIALS AND METHODS

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Propolis. Propolis samples were obtained from Wolgast Tree Farm and Apiary located in a

79

suburban area of central New Jersey (Somerset, NJ, USA). Upon arrival samples were stored in

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air tight glass containers in a –80 °C freezer prior to analysis. For cAEDA and identification

81

experiments, the propolis samples were collected from a single beehive during the spring season

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of three consecutive years (2011 (year 1); 2012 (year 2); and 2013 (year 3)). For the SIDAs and

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odor simulation experiments, the propolis samples were collected from a single beehive during

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the spring season of 2015.



85

Solvents. Chromatographic grade diethyl ether was obtained from Honeywell Burdick &

86

Jackson (Muskegon, MI, USA) and freshly distilled in-house prior to use. Pentane was obtained

87

from Fisher Scientific (Pittsburgh, PA, USA) and was freshly distilled in-house prior to use.

88

Reference Compounds. The following compounds were obtained from commercial suppliers

89

given in parentheses: 1–19, 21–27, 30–38, 40–43, 45–48, (2H6)benzene, (2H8)toluene, and

90

(2H8)naphthalene (Sigma Aldrich, St. Louis, MO, USA); 20 & 44 (Penta Manufacturing

91

Company, Livingston, NJ, USA); 28 & 39 (Vigon International, East Stroudsburg, PA, USA).

92

The trans-4,5-epoxy-(2E)-dec-2-enal was synthesized as described in the literature.24

93

Isotopically Substituted Odorants. (2H3)-3, (2H6)-5, (2H2)-11, (2H5)-12, (13C2)-14, (13C2)-15,

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(2H5)-16, (2H9)-18, (2H5)-19, (13C2)-20, (2H4)-22, (13C)-23, (2H3)-24, (2H4)-26, (2H3)-27, (2H7)-34,

95

(2H3)-37, (2H5)-38, (13C)-40, (2H3)-42, (13C)-45, (13C)-47, (2H2)-48 were purchased from

96

aromaLAB (Planegg, Germany). (2H7)-32 and (2H7)-39 were not commercially available and were

97

therefore synthesized in-house.

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Synthesis of (2H7)cinnamaldehyde. Lithium aluminum hydride (763 mg, 20 mmol) was added

99

slowly to a solution of (2H7)cinnamic acid (310 mg, 2 mmol) (Sigma Aldrich, St. Louis, MO, USA)

100

in anhydrous diethyl ether (20 mL) under nitrogen at room temperature. The mixture was stirred

101

for one hour, at which point deionized water was added until no more gas was produced. To the

102

resulting solution, sulfuric acid (2M) was added until the precipitate was dissolved. The organic

103

phase was collected, and the aqueous phase was extracted with diethyl ether (3 × 20 mL). The

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organic fractions were combined and evaporated under reduced pressure until (2H7)cinnamyl

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alcohol was obtained as a clear solid (170 mg, 1.2 mmol, 60% yield). To initiate the synthesis of

106

(2H7)cinnamaldehyde, pyridinium chlorochromate (516 mg, 2.4 mmol) and sodium acetate (480

107

mg, 5.85 mmol) were added to a solution of (2H7)cinnamyl alcohol (160 mg, 1.1 mmol) in


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dichloromethane (40 mL) under nitrogen. The liquid was refluxed for two hours, then filtered

109

through celite under vacuum. The filtrate was dried over sodium sulfate, then placed into a

110

dropping funnel of a SAFE apparatus. The SAFE was thermostated at 40 °C and kept under high

111

vacuum (10-3 Pa). The SAFE distillate was thawed to room temperature and the concentration of

112

the target compound was determined by GC-FID using isotopically unmodified cinnamaldehyde

113

as a reference standard. The synthesis of (2H7)cinnamaldehyde was confirmed by GC-MS (Figure

114

1).

115

Synthesis of (2H7)cinnamyl acetate. (2H7)cinnamyl alcohol (10 uL), synthesized as described

116

above, as well as acetic anhydride (10 uL) and 4-(dimethylamino)pyridine (DMAP, 0.5 mg) were

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added to a solution of anhydrous pyridine (1 mL) under nitrogen at room temperature, then stored

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at 0 °C overnight. To the solution, diethyl ether (20 mL) was added. The solution was placed on

119

an autoshaker for 10 minutes, then put into a dropping funnel of a SAFE apparatus. The SAFE

120

was thermostated at 40 °C and kept under high vacuum (10-3 Pa). The SAFE distillate was thawed

121

to room temperature and the concentration of the target compound was determined by GC-FID

122

using isotopically unmodified cinnamyl acetate as a reference standard. The synthesis of

123

(2H7)cinnamyl acetate was confirmed by GC-MS (Figure 2).

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Sensory Analyses. Free choice profiling. Free choice profiling was performed to determine the

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sensory lexicon of the propolis samples. Propolis samples (1 g) were crushed into small pieces and

126

placed in 20 mL glass scintillation vials (Thermo Fisher Scientific, Fair Lawn, NJ, USA). The

127

sensory evaluation was conducted by seven experienced panelists who performed sensory

128

evaluation on a day-to-day basis. Each sample was given to the sensory panel for free-choice

129

profiling. The panelists were asked to smell the propolis samples (one at a time) and to use their

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own terms to describe the odor of the given sample. This test was executed on each sample



131

(propolis years 1, 2, and 3). All of the descriptors were pooled and the most common descriptors

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were selected for a sensory evaluation by quantitative olfactory profile analysis.

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Quantitative olfactory profile analysis. In this test performed by seven trained panelists, the

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propolis samples (1 g) were placed in capped glass scintillation vials and provided to the panelists

135

for orthonasal evaluation. Each of the samples (propolis years 1, 2, and 3), and reference

136

compounds dissolved in water were presented to the sensory panel at the same time. All samples

137

from each year were stored in -80 °C until the analysis was performed. Samples were evaluated in

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triplicate and sample presentation order randomized. The eight reference compounds used in this

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stage of the study were selected according to the free-choice profile results. The reference

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compounds included (3E)-hex-3-enal (green), butanoic acid (cheesy, sweaty), phenylacetaldehyde

141

(floral), ethyl cinnamate (cinnamon), 1,8-cineole (eucalyptus), eugenol (clove), β-pinene (piney),

142

and phenylacetic acid (honey). All reference solutions were prepared in water at 100 times above

143

their individual thresholds. The panelists were asked to individually remove the caps of the

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scintillation vials and evaluate the intensity of the given odor qualities using a 7-point category

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scale, ranging from 0 (not observable) to 3 (strong observable).

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Preparation of Volatile Isolates. A small amount of propolis (~5.5 g) was frozen in liquid

147

nitrogen, and ground to a fine powder with a laboratory mill. An exact amount of the powder (5

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g) was transferred to a centrifuge tube, and freshly distilled diethyl ether (100 ml) was added. The

149

sample was placed on an auto-shaker (Burrell Wrist Action Shaker) for 15 min. For separation of

150

the solvent extract from solid residue, the sample was centrifuged for 15 min at 4500 rpm. Solvent-

151

assisted flavor evaporation (SAFE) was employed to separate volatile from non-volatile

152

compounds. The SAFE distillate was dried over anhydrous sodium sulfate, filtered, and

153

concentrated to ~2 mL using a Vigreux column. Finally, the volatile isolate was concentrated to


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~200 µL with a gentle stream of nitrogen gas, and then placed into a GC vial with insert for GC

155

analysis.

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Gas Chromatography-Mass Spectrometry (GC-MS). GC-MS was performed on an Agilent

157

6890 series gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to an

158

Agilent 5973 mass spectrometer detector. The capillary column used for chromatographic

159

separation was a fused silica GC column HP-FFAP, 30 meters in length, with a 0.25 mm inner

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diameter and a 0.25 µm film thickness (30 m × 0.25 mm × 0.25 µm) (Agilent). An on-column

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injection of the propolis volatile isolate (1 µL) was made by an autosampler with a 10 µL syringe.

162

Helium was used as a carrier gas with a constant flow of 1 mL/min. The oven temperature was

163

initially held at 35 °C for 1 minute followed by an increase in temperature at a rate of 60 °C/min

164

until the oven temperature reached 60 °C. Subsequently, the oven was heated at a rate of 6 °C/min

165

to reach 250 °C and held at this temperature for 5 minutes. The mass spectrometer detector was

166

coupled to the GC via a transfer line heated at 250 °C and operated in electron ionization (EI)

167

mode at 70 eV. The detector scan range was m/z 50–350.

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Purge and Trap Thermal Desorption Gas Chromatography Mass Spectrometry (P&T-TD-

169

GC-MS). A Solid Sample Purge & Trap Oven Collection System (Model #782050 Scientific

170

Instrument Services, Ringoes, NJ) was used for purge and trap collection of volatiles. All three

171

samples were crushed into a fine powder, and ~100 mg of each sample powder was measured into

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a 14-inch bosrosilicate glass thermal desorption tube (0.5 in o.d. by 0.36 in i.d.). Glass wool (0.5

173

g) was plugged into each glass tube from both ends. The tubes were placed in the sample collection

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oven and connected from one end (exhaust) with a glass-lined stainless-steel (GLT) thermal

175

desorption trap tube packed with Tenax TM (6 cm long × 3 mm i.d. tube) as an adsorbent trap, and

176

the other end to a gas supply that purged nitrogen gas with a flow rate of 50 mL/min. Before



177

desorption, the trap tubes were spiked with (2H6)benzene, (2H8)toluene, and (2H8)naphthalene (1

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µg each) as internal standards. The glass tubes were then incubated (100 °C) in the sample

179

collection oven for 30 min.

180

For analysis, the traps were connected to a Short Path Thermal Desorption Unit, Model

181

TD-2 (Scientific Instrument Services, Ringoes, NJ) located on the top of the GC injection port.

182

The attached trap tube was purged with helium gas for 10 seconds to flush the trap and needle of

183

air (oxygen) prior to injection. Samples were then thermally desorbed from the trap into the GC

184

injection port at 250 °C for 5 min.

185

A Varian 3400 GC system coupled to Finnigan MAT8230 double focusing magnetic sector

186

MS was used for these analyses. The GC was equipped with ZB-5 capillary column (60 m × 0.32

187

mm i.d. × 1.0 µm film thickness) (Phenomenex, Torrance, CA). Helium was used as carrier gas

188

with a flow rate of 1.0 mL/min. The injection was done in a split ratio of 10:1. The initial GC

189

temperature (–20 °C) was held with dry ice for 5 minutes to ensure cryofocusing, after which the

190

temperature was increased to 280 °C at a rate of 10 °C/min. The GC-MS interface line was held at

191

280 °C. The mass spectrometer was in EI (70 eV) mode with ion source temperature at 250 °C.

192

Scans were made from 35 m/z to 350 m/z, scan time was 0.6 s, and interscan time was 0.8 s.

193

Gas Chromatography-Olfactometry (GC-O). An Agilent Technologies Gas Chromatograph

194

6890 series with FID detector was employed. A 10 µL syringe was used to manually inject the

195

sample (1 µL). Cold on-column sample injection was performed at an initial temperature of 35 °C.

196

Helium was used as a carrier gas with a flow rate of 1.5 mL/min. The initial temperature was held

197

for 1 min, then increased to 60 °C at a rate of 60 °C/min. After reaching 60 °C, the temperature

198

was increased at 6 °C/min until it reached 240 °C, and held for 10 min. DB-5 and HP-FFAP

199

capillary columns (30 m × 0.32 mm × 0.25 µm) were used for chromatographic separation. A Y-


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type splitter was placed at the end of capillary column and divided the effluent at a 1:1 volume

201

ratio into two 50 cm long sections of uncoated fused silica capillaries. One portion of the effluent

202

was routed to the flame ionization detector (FID) while the other portion was channeled into the

203

sniffing port. The FID detector was held at 250 °C with a hydrogen flow of 40.0 mL/min and air

204

flow of 450 mL/min. Helium was used as a make-up gas with a constant flow of 45 mL/min. A

205

custom-machined cylindrical cone (80 mm × 25 mm I.D.) sniffing port was installed in the heating

206

block of the front of the FID detector and heated to 180 °C. Each isolate was evaluated by two

207

panelists and FD factors determined by the panelist were reported as averages.

208

Comparative Aroma Extract Dilution Analysis (cAEDA). The propolis volatile isolates were

209

diluted with diethyl ether (1:2 by vol.) resulting in a series of dilutions of 1:2, 1:4, 1:8, 1:16, 1:32,

210

1:64, 1:128, 1:256, 1:512, and 1:1024. Each diluted sample was analyzed by GC-O on the FFAP

211

column according to the conditions described above. Flavor dilution (FD) factors within a range

212

of FD 2–FD 1024 were assigned to all odorants detected during GC-O, indicating the highest

213

dilution at which the odorant was detected.

214

Fractionation of the Propolis Volatile Isolate by Solid Phase Extraction (SPE). A volatile

215

isolate was prepared as described above using pentane as solvent. Prior to introducing the sample,

216

the silica gel SPE cartridge (2 g/12 mL Giga Tube, Strata SI-1 Silica (55 µm, 70Å)) (Phenomenex,

217

Torrance, CA) was sequentially conditioned with solvents: pentane (100%), diethyl ether (100%)

218

and pentane (100%) (5 mL each). The volatile isolate was loaded on the SPE cartridge, connected

219

to a manifold, and fractionation was performed under vacuum. Elution was performed by pentane

220

(100%), fraction A; pentane/diethyl ether (98:2 v:v), fraction B; pentane/diethyl ether (95:5 v:v),

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fraction C; pentane/diethyl ether (90:10 v:v), fraction D; pentane/diethyl ether (50:50 v:v), fraction

222

E; diethyl ether (100%), fraction F (5 mL each). Fractions A–F were concentrated to ~200 µL



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under a gentle stream of nitrogen prior to GC analysis. The fraction used for identification of each

224

odorant can be found in Table 3.

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Stable Isotope Dilution Assays (SIDAs). Freshly distilled diethyl ether (100 ml) was combined

226

with a sample of frozen and ground propolis (5 g) in a 250 mL centrifuge tube. Isotopically

227

substituted analogous to each of the target compounds were spiked into the mixture as internal

228

standards prior to extraction. Extraction, SAFE, and subsequent concentration were carried out as

229

detailed above. The concentration of each target compound was calculated in µg/kg from the

230

analyte peak area, the standard peak area, the amount of propolis sample used, the amount of

231

standard added and a response factor which was previously determined. Peak areas were collected

232

from extracted ion chromatograms (EIC) using m/z values characteristic for the analyte and the

233

standard. For each analyte, the m/z values (analyte/standard) and response factor (RF) used can be

234

found in Table 1. All values were reported as the mean of at least duplicate measurements using

235

Microsoft Excel for Office 365 MSO version 1811 (Microsoft Corporation, Redman, WA).

236

Quantitation of 2-Methylbutanoic Acid (17) and 3-Methylbutanoic Acid (18).

237

concentrations of 17 and 18 were calculated using a slightly modified method as previously

238

reported.25 Mixtures of 17 and 18 (5 in total; 1:0, 3:1, 1:1, 1:3, 0:1) were analyzed by GC-MS and

239

a regression equation was calculated using the area ratio of m/z 60 by the sum of m/z 60 + m/z 74

240

against the percentage of 18 in the mixture. The ratio of 17 and 18 was then determined in the

241

propolis sample using the equation. Subsequently, the concentration of the total mixture of 17 and

242

18 was quantified in the propolis sample by SIDA using (2H9)-18 (m/z 60/63, RF 0.91). The

243

individual concentrations of 17 and 18 were then determined by taking the concentration of the

244

sum of 17 and 18 and calculating the level of each isomer based on the ratio in the sample.


The

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Odor Thresholds. To calculate OAVs for each of the odorants that were quantitated through

246

SIDA, orthonasal odor thresholds were determined in low odor sunflower oil as lipophilic model

247

matrix. Threshold determinations were carried out at the Leibniz-Institute for Food Systems

248

Biology at the Technical University using the procedure for the determination of odor and taste

249

thresholds by a forced-choice ascending concentration series method of limits published by the

250

American Society for Testing and Materials (ASTM).26

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RESULTS AND DISCUSSION

252

Sensory Characterization. Propolis samples (3 in total) collected over three consecutive seasons

253

(year 1, year 2, and year 3) were sensorially evaluated by trained panelists, using free-choice

254

profiling (Table 2) followed by a quantitative olfactory profile analysis (Figure 3). The olfactory

255

profiles were similar for each of the three years, all displaying odor characters similar to propolis.

256

However, there were some differences exhibited amongst samples. The olfactory profile of the

257

propolis sample in year 1 displayed a strong propolis odor with clove, honey, and cinnamon notes,

258

as well as some floral, caramel, green, and cheesy notes. The olfactory profile of the year 2 propolis

259

sample showed the lowest overall intensity of all the three years sampled. It was characterized by

260

a typical propolis attributes with clove, honey, and cinnamon notes, but with less intense floral,

261

eucalyptus, and piney notes. The olfactory profile of the year 3 propolis sample was the most

262

similar to that of year 1, displaying a strong propolis odor character with clove, cinnamon and

263

honey notes, and some piney, eucalyptus, and floral notes. In summary, all three years had a similar

264

overall olfactory profile with propolis samples of years 1 and 3 being the most similar and the

265

propolis sample of year 2 having the mildest odor.

266

Odorant Screening. The three propolis samples, collected over three consecutive seasons, were

267

individually ground into a fine powder and extracted with diethyl ether. Extracts were subjected



268

to SAFE and distillates were concentrated. When evaluated sensorially, the volatile isolates

269

reflected the olfactory profiles the original propolis samples, particularly their character was

270

consistent with the sensory differences between the samples of the individual years. The propolis

271

volatile isolates were subjected to cAEDA to determine the odor-active compounds present in each

272

sample. To aid in identification of some odorants that were detected during GC-O analysis but had

273

either very low MS signals or co-eluted with other compounds during chromatography, additional

274

techniques were employed. Specifically, P&T-TD-GC-MS was used to identify compounds that

275

co-eluted with diethyl ether and SAFE isolates were fractionated by SPE before being re-analyzed

276

by GC-O and GC-MS. Three odorants, namely, 1-octen-3-one, 2,3-diethyl-5-methylpyrazine, and

277

trans-4,5-epoxy-(2E)-dec-2-enal, occurred at too low of a concentration in the propolis isolates to

278

acquire MS spectra; however, they were unequivocally identified by comparing their odor quality

279

and intensity, and their RI (on both FFAP and DB-5 columns) with that of authentic reference

280

odorants. In summary, a total of 48 odorants were identified with FD factors ranging from 4 to

281

1024 (Table 3). To our knowledge, of these 48 compounds, 21 have not been previously identified

282

as volatiles nor odor-active compounds in propolis.

283

cAEDA and subsequent identification experiments resulted in the identification of a total

284

of 13 odorants which exhibited high FD factors ≥ 256 in a sample from at least one of the years

285

(Table 3). The highest FD factors were determined for 2-methoxy-4-vinylphenol (clove), -pinene

286

(piney), 1,8-cineole (eucalyptus), (2E)-non-2-enal (green), (2E,6Z)-nona-2,6-dienal (cucumber),

287

2-methoxyphenol (smoky), 2-phenylethanol (floral, rose), -ionone (floral, violet), eugenol

288

(clove), (E)--damascenone (cooked apple), cinnamaldehyde (cinnamon), ethyl cinnamate

289

(cinnamon), and 3-phenylpropanoic acid (floral). Among the odorants, clove smelling 2-methoxy-

290

4-vinylphenol (42) displayed a FD factor ≥ 1024 in all three seasons. Although 42 was previously


Page 14 of 34

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291

reported as a volatile in honey, to date it has not been described as an odor-active compound in

292

propolis.19, 25, 27 The sensory results indicated that propolis sample year 1 was more similar to

293

propolis sample year 3. Particularly propolis samples years 1 and 3 were higher in honey, clove,

294

green, and floral notes than propolis year 2. The similar clove odor intensity of propolis samples

295

from year 1 and 3 may be explained by similar FD factors for the clove odorant eugenol (40, years

296

1 and 3; FD 256) as compared to year 2 (40, FD 64). Similarly, the honey and floral odor intensities

297

of the propolis samples from years 1 and 3 may be explained by similar FD factors for the odorants

298

phenylacetaldehyde (floral, honey) (16, years 1 and 3; FD 64), phenylacetic acid (honey) (46, years

299

1 and 3; FD 16) and 3-phenylpropanoic acid (floral) (48, years 1 and 3; FD 256) as compared to

300

year 2, (16, FD 16), (46, FD 4), and (48, FD 64) respectively. Although propolis samples in year

301

1 and 3 were similar in odor profile, propolis in year 3 had higher intensities of piney, eucalyptus

302

and cinnamon notes than propolis year 1. The higher intensity of these notes in propolis year 3 as

303

compared to propolis years 1 and 2 may be explained by the higher FD factor of the odorants, -

304

pinene (3, FD 256), 1,8 cineole (5, FD 256), and ethyl cinnamate (38, FD 256) and ethyl-3-

305

phenylpropanoate (25, FD 64) in the year 3 sample. Although the overall difference between years

306

was small, some of the sensory distinctions observed in this study may be explained by the variable

307

FD factors observed for selected odorants during the cAEDA.

308

Of the total 48 odorants identified in the propolis samples, 40 were detected in all propolis

309

samples (years 1 through 3). The earthy smelling odorant 2,3-diethyl-5-methylpyrazine was

310

detected only in propolis year 1, and the coconut smelling and floral / honey smelling odorants, -

311

octalactone and phenylethyl acetate, were detected only in propolis year 3. In addition, five

312

odorants, namely, hexanal, (3E)-hex-3-enal, -terpinene, 1-octen-3-one, and -nonalactone, were

313

detected only in propolis years 1 and 3 and not in propolis year 2. Nevertheless, the propolis



314

samples were remarkably consistent over the three consecutive seasons, resulting in a total of 40

315

odorants detected in all three years with similar FD values amongst the three seasons.

316

OAVs of Propolis Odorants. Based on the results of the cAEDA, 13 odorants, namely,

317

(2E,6Z)-nona-2,6-dienal, ethyl cinnamate, (2E)-non-2-enal, 2-methoxy-4-vinylphenol, α-pinene,

318

1,8-cineole, 2-methoxyphenol, cinnamaldehyde, (E)-β-damascenone, 2-phenylethanol, β-ionone,

319

eugenol, and 3-phenylpropanoic acid exhibited an FD factor ≥ 256 in a sample from at least one

320

of the seasons (Table 3). Therefore, these odorants were chosen for quantitation in a propolis

321

sample collected from the same beehive in the summer of 2015. Based on the results of the SIDA,

322

OAVs were calculated for the odorants (Table 4) and an odor model was prepared in deodorized

323

beeswax for the 10 compounds with FD ≥ 256 and OAV ≥ 1 (odor model 1; Figure 4). When

324

compared sensorially to the authentic propolis sample, the model was clearly reminiscent of

325

propolis; however, it was not a satisfactory match due to higher honey and caramel notes and lower

326

green and cheesy notes. To be able to more closely simulate the odor of propolis, an additional set

327

of 13 odorants with FD factors ≥ 64 were quantitated, namely, γ-decalactone, (2E,4E)-nona-2,4-

328

dienal, methyl cinnamate, 3-methylnonane-2,4-dione, cinnamyl acetate, hexanoic acid, 2-

329

methylbutanoic acid, phenylacetaldehyde, vanillin, 3-methylbutanoic acid, butanoic acid, linalool,

330

and (E)-isoeugenol. Based on the SIDA results, OAVs were calculated for the additional set of

331

odorants, resulting in a total of 22 compounds with an OAV ≥ 1. When the 22 odorants were

332

combined in the deodorized beeswax matrix (odor model 2; Figure 4), the model closely matched

333

the sensory characteristics of the authentic propolis sample. Four compounds included in odor

334

model 2, namely, butanoic acid, 3-methylbutanoic acid, 2-methylbutanoic acid and hexanoic acid,

335

all with an FD factor of 64, exhibit rancid and sweaty odor characters. These compounds may be

336

responsible for elevating the cheese odor note of odor model 2 as compared to odor model 1.


Page 16 of 34

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337

Additionally, (E)-isoeugenol (OAV 3700) and linalool (OAV 380) showed the highest OAVs

338

calculated for all 22 compounds, therefore these compounds may also contribute to the

339

improvement of the model.

340

Although a high FD factor of 1024 was observed for 2-methoxy-4-vinylphenol in all

341

samples (years 1 through 3), a relatively low OAV was calculated for the compound (OAV 26).

342

This may be due, in part, to a matrix effect in beeswax and an overestimation of its odor potency

343

by AEDA. Additionally, the three compounds with the highest OAVs, (E)-isoeugenol, linalool and

344

butanoic acid (FD 64) did not display the highest FD factors determined by AEDA. During AEDA,

345

odorants are perceived through the sniffing port and their perception are dependent upon their odor

346

threshold in air, which may result in an over- or underestimation of their impact in the natural

347

matrix, thus supporting the importance of SIDA quantitation, OAV calculation, and odor

348

simulation experiments to discover the key odorants. Studies on the matrix effect of odorant release

349

from beeswax and omission experiments to elucidate the contribution of the individual odorants

350

to the overall olfactory profile are currently underway and will be published separately.

351

In summary, the results of the present investigation revealed that propolis collected from

352

the same beehive over three consecutive seasons had similar overall olfactory profiles with only

353

slight differences in odor intensity. Propolis samples of years 1 and 3 being the most similar in

354

odor intensity and the propolis sample of year 2 having the mildest odor. Application of cAEDA

355

resulted in the identification of 48 odorants of which 40 were detected in all three years, including

356

21 compounds, that have not been previously reported as odorants in propolis. Furthermore, the

357

quantitation of the odorants by SIDA, calculation OAVs, and odor reconstitution experiments

358

revealed that the odor profile of propolis can be closely simulated by a mixture of 22 odorants

359

when combined at their natural concentrations. The results of the present investigation will help to



360

establish a basis for future research on the variability of propolis sourced from different

361

geographical locations, produced by different bee species, and collected from different botanical

362

sources (i.e., the propolis foraging behavior of bees), all of which is largely unknown. In addition,

363

this present work can be used to aid the development of propolis odor standardization and quality

364

control methods, which currently represent a major gap in the food, beverage, and consumer goods

365

industry.

366 367

ACKNOWLEDGMENTS

368

This work was supported by the USDA National Institute of Food and Agriculture Hatch Project

369

#1016031.

370 371

AUTHOR INFORMATION

372

Corresponding Author

373

*(J.M) E-mail: [email protected]. Fax: 865-974-7332. Phone: 865-974-7247.

374

Notes

375

The authors declare no competing financial interest.

376

ABBREVIATIONS USED

377

AEDA, aroma extract dilution analysis; ARS, Agricultural Research Service; cAEDA,

378

comparative aroma extract dilution analysis; FD factor, flavor dilution factor; GC-O, gas

379

chromatography−olfactometry; SAFE, solvent-assisted flavor evaporation; SIDA, stable isotope

380

dilution assay; USDA, United States Department of Agriculture.

381

Nomenclature


Page 18 of 34

Page 19 of 34


382

1,8-cineole, 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane; cinnamaldehyde, (2E)-3-phenylprop-2-

383

enal; cinnamyl alcohol, (2E)-3-phenylprop-2-en-1-ol; cinnamyl formate, (2E)-3-phenylprop-2-en-

384

1-yl formate; (E)-β-damascenone, (2E)-1-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)but-2-en-1-

385

one; γ-decalactone, 5-hexyltetrahydrofuran-2-one; trans-4,5-epoxy-(2E)-dec-2-enal, (2E)-3-

386

[(2R,3R)/(2S,3S)-3-butyloxiran-2-yl]prop-2-enal; ethyl cinnamate, ethyl (2E)-3-phenylprop-2-

387

enate; eugenol, 2-methoxy-4-(prop-2-en-1-yl)phenol; HDMF, 4-hydroxy-2,5-dimethylfuran-

388

3(2H)-one; β-ionone, (3E)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one; (E)-isoeugenol,

389

2-methoxy-4-[(1E)-prop-1-en-1-yl]phenol;

390

cinnamate, methyl (2E)-3-phenylprop-2-enoate; γ-nonalactone, 5-pentyltetrahydrofuran-2-one; δ-

391

octalactone, 6-propyltetrahydropyran-2-one; α-pinene, 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene; γ-

392

terpinene,

393

methoxybenzaldehyde;

linalool,

3,7-dimethylocta-1,6-dien-3-ol;

1-methyl-4-(propan-2-yl)cyclohexa-1,4-diene;


vanillin,

methyl

4-hydroxy-3-


REFERENCES 1. Seeley, T.; Morse, R. The nest of the honey bee (Apis mellifera L.). Insectes Soc. 1976, 23, 495–512. 2. Fearnley, J. Bee propolis: natural healing from the hive; Souvenir Press: London, England, 2001. 3. Herbert, E. The hive and the honey bee; Graham, J., Ed.; Dadant & Sons: Hamilton, IL, 1992. 4. Marinescu, I.; Tamas, M. Poplar buds-a source of propolis. Apiacta 1980, 15, 121–126. 5. Wollenweber, E.; Asakawa, Y.; Schillo, D.; Lehmann, U.; Weigel, H. A novel caffeic acid derivative and other constituents of Populus bud excretion and propolis (bee-glue). Z. Naturforsch. C Bio. Sci. 1987, 42, 1030–1034. 6. Shimanuki, H.; Flottum, K.; Harman, A. The ABC & XYZ of bee culture: an encyclopedia pertaining to the scientific and practical culture of honey bee, 41st Ed.; The AI Root Company: Medina, OH, 2007. 7. Haydak, M. Propolis. Report of the Iowa State Apiarist for 1953: 74–87. 8. Castaldo, S.; Capasso, F. Propolis, an old remedy used in modern medicine. Fitoterapia 2002, 73, S1–S6. 9. Grange, J.; Davey, R. Antibacterial properties of propolis (bee glue). J. R. Soc. Med. 1990, 83, 159. 10. Kimoto, T.; Aga, M.; Hino, K.; Koya-Miyata, S.; Yamamoto, Y.; Micallef, M. J.; Hanaya, T.; Arai, S.; Ikeda, M.; Kurimoto, M. Apoptosis of human leukemia cells induced by Artepillin C, an active ingredient of Brazilian propolis. Anticancer Res. 2001, 21, 221–228.


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11. Takagi, Y.; Choi, I.-S.; Yamashita, T.; Nakamura, T.; Suzuki, I.; Hasegawa, T.; Oshima, M.; Gu, Y.-H. Immune activation and radioprotection by propolis. Am. J. Chin. Med. 2005, 33, 231– 240. 12. Chan, G. C.-F.; Cheung, K.-W.; Sze, D. M.-Y. The immunomodulatory and anticancer properties of propolis. Clin. Rev. Allergy Immunol. 2013, 44, 262–273. 13. Bunney, M. H. Contact dermatitis in beekeepers due to propolis (bee glue). Br. J. Dermatol. 1968, 80, 17–23. 14. Yang, C.; Luo, L.; Zhang, H.; Yang, X.; Lv, Y.; Song, H. Common aroma‐active components of propolis from 23 regions of China. J. Sci. Food Agric. 2010, 90, 1268–1282. 15. Melliou, E.; Stratis, E.; Chinou, I. Volatile constituents of propolis from various regions of Greece–Antimicrobial activity. Food Chem. 2007, 103, 375–380. 16. Torres, R. N. S.; Lopes, J. A. D.; Neto, J. M. M.; Citó, A. Constituintes voláteis de própolis piauiense. Quím. nova 2008, 31, 479–485. 17. Markham, K. R.; Mitchell, K. A.; Wilkins, A. L.; Daldy, J. A.; Lu, Y. HPLC and GC-MS identification of the major organic constituents in New Zeland propolis. Phytochemistry 1996, 42, 205–211. 18. Greenaway, W.; Scaysbrook, T.; Whatley, F. R. The analysis of bud exudate of Populus x euramericana, and of propolis, by gas chromatography–mass spectrometry. Proc. R. Soc. Lond. B 1987, 232, 249–272. 19. Silici, S.; Kutluca, S. Chemical composition and antibacterial activity of propolis collected by three different races of honeybees in the same region. J. Ethnopharmacol. 2005, 99, 69–73.



20. Righi, A. A.; Alves, T. R.; Negri, G.; Marques, L. M.; Breyer, H.; Salatino, A. Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities. J. Sci. Food Agric. 2011, 91, 2363–2370. 21. Bankova, V.; Christov, R.; Kujumgiev, A.; Marcucci, M.; Popov, S. Chemical composition and antibacterial activity of Brazilian propolis. Z. Naturforsch. C Bio. Sci. 1995, 50, 167–172. 22. Greenaway, W.; May, J.; Scaysbrook, T.; Whatley, F. Identification by gas chromatographymass spectrometry of 150 compounds in propolis. Z. Naturforsch. C Bio. Sci. 1991, 46, 111–121. 23. Segueni, N.; Khadraoui, F.; Moussaoui, F.; Zellagui, A.; Gherraf, N.; Lahouel, M.; Rhouati, S. Volatile constituents of Algerian propolis. Ann. Biol. Res. 2010, 1, 103–7. 24. Gassenmeier, K.; Schieberle, P. Formation of the intense flavor compoundtrans-4, 5-epoxy(E)-2-decenal in thermally treated fats. J. Am. Oil Chem.’ Soc. 1994, 71, 1315–1319. 25. Ruisinger, B.; Schieberle, P. Characterization of the key aroma compounds in rape honey by means of the molecular sensory science concept. J. Agric. Food Chem. 2012, 60, 4186–4194. 26. American Society of Testing and Materials. Standard E679-04. Standard practice for determination of odor and taste thresholds by a forced-choice ascending concentration series method of limits. In ASTM Book of Standards; American Society of Testing and Materials: West Conshohocken, PA, USA, 2005 Vol. 15.08, pp 38–44. 27. Uzel, A.; Önçağ, Ö.; Çoğulu, D.; Gençay, Ö. Chemical compositions and antimicrobial activities of four different Anatolian propolis samples. Microbiol. Res. 2005, 160, 189–195. 28. Torres, R. N. S.; Lopes, J. A. D.; Neto, J. M. M.; Citó, A. Constituintes voláteis de própolis piauiense. Quím. Nova 2008, 31, 479–485.


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Table 1. Response Factors and Ions Selected for Odorants and Labeled Isotopes Used for Stable Isotope Dilution Analysis

no. odorant 3 -pinene 5 1,8-cineole 11 (2E)-non-2-enal 12 linalool 14 (2E,6Z)-nona-2,6-dienal 15 butanoic acid 16 phenylacetaldehyde 18 3-methylbutanoic acid 19 (2E,4E)-nona-2,4-dienal 20 3-methylnonan-2,4-dione 22 (E)--damascenone 23 hexanoic acid 24 2-methoxyphenol 26 2-phenylethanol 27 -ionone 32 cinnamaldehyde 34 methyl cinnamate 37 γ-decalactone 38 ethyl cinnamate 39 cinnamyl acetate 40 eugenol 42 2-methoxy-4-vinylphenol 45 (E)-isoeugenol

labelled standard -pinene-d3 1,8-cineole-d6 (2E)-non-2-enal-d2 linalool-d5 (2E,6Z)-nona-2,6-dienal-13C2 butanoic acid-13C2 phenylacetaldehyde-d5 3-methylbutanoic acid-d9 (2E,4E)-nona-2,4-dienal-d5 3-methylnonan-2,4-dione-13C2 (E)--damascenone-d4 hexanoic acid-13C 2-methoxyphenol-d3 2-phenylethanol-d4 -ionone-d3 cinnamaldehyde-d7 methyl cinnamate-d7 γ-decalactone-d3 ethyl cinnamate-d5 cinnamyl acetate-d7 eugenol-13C 2-methoxy-4-vinylphenol-d3 (E)-isoeugenol-13C

47 vanillin 48 3-phenylpropanoic acid

vanillin-13C 3-phenylpropanoic acid-d2


Ion (m/z) analyte standard

RF

93 154 83 93 70 60 91 60 138 170 69 60 124 91 177 131 131 85 176 176 164 150 164

96 160 85 98 72 62 95 63 143 174 73 61 127 94 180 137 138 86 181 183 165 153 165

0.74 1.03 0.69 0.66 0.80 1.03 1.07 0.91 0.99 0.99 0.70 0.78 0.93 0.92 0.63 0.32 0.65 0.76 1.13 1.00 1.12 1.00 0.89

123 150

124 152

1.09 1.00


Table 2. Sensory Characterization of three Propolis Samples using Descriptors Determined by Orthonasal Free-Choice Profiling. propolis year 1 year 2 year 3

odor description strong propolis odor with clove, honey, and cinnamon notes, and some floral, caramel, green and cheesy notes mildest propolis odor with clove, honey, and cinnamon notes strong propolis odor with clove, cinnamon, and honey notes and some piney, eucalyptus, and floral notes


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Table 3. Propolis Odorants (FD ≥ 4) in Samples Collected over three Consecutive Years (Year 1 through 3). no.a

odorantb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

hexanalj (3E)-hex-3-enalj -pinenej -terpinene 1,8-cineole 3-methylbutan-1-olj 1-octen-3-oneh dimethyl trisulfide acetic acidj 2,3-diethyl-5-methylpyrazineh (2E)-non-2-enal linaloolj 2-methylpropanoic acidj (2E,6Z)-nona-2,6-dienal butanoic acidj phenylacetaldehydej 2-methylbutanoic acidij 3-methylbutanoic acidij (2E,4E)-nona-2,4-dienal 3-methylnonan-2,4-dione phenylethyl acetate (E)--damascenone hexanoic acid 2-methoxyphenol ethyl 3-phenylpropanoate 2-phenylethanolj -ionone δ-octalactone trans-4,5-epoxy-(2E)-dec-2-enalh γ-nonalactone HDMF cinnamaldehydej 4-methoxybenzaldehydej methyl cinnamatej cinnamyl formate 4-methylphenol γ-decalactone ethyl cinnamate cinnamyl acetate eugenolj 4-ethylphenol 2-methoxy-4-vinylphenol 2,6 dimethoxyphenol cinnamyl alcoholj (E)-isoeugenol phenylacetic acid vanillinj 3-phenylpropanoic acid

odor green green piney terpene eucalyptus malty mushroom cabbage vinegar earthy green floral sweaty, rancid cucumber sweaty, rancid floral, honey

RId on FFAP DB-5 1085 801 1130 792 1133 939 1185 979 1194 1014 1200 977 1285 980 1385 968 1420 600 1495 1158 1530 1161 1550 1096 1565 1215 1580 1150 1610 772 1639 1045

}sweaty, rancid

1661

885

64

16

16

F

fatty hay floral cooked apple rancid smoky cinnamon floral, rose floral, violet coconut metallic coconut caramel cinnamon anise cinnamon cinnamon barnyard coconut cinnamon cinnamon clove phenolic clove smoky floral clove honey vanilla floral

1698 1715 1785 1810 1840 1860 1888 1901 1980 1963 2000 2020 2040 2044 2050 2053 2065 2089 2125 2130 2144 2177 2168 2248 2271 2284 2350 2558 2600 2620

1212 1246 1193 1384 973 1087 1390 1108 1488 1278 1380 1361 1080 1267 1299 1300 1332 1178 1466 1467 1389 1359 1178 1313 1349 1304 1451 1274 1394 1343

64 64

4 16

E F E

256 64 64 16 256 256

64 16 256 16 64 64

4 16 16 256 16 64 16 4 64 64 64 256 4 1024 16 16 16 16 64 256

4

16 64 4 64 16 16 64 64 64 16 4 16 16 64 4 64 16 4 64 256 16 256 4 1024 16 16 64 16 16 256

qualityc


FDe factor year 1 year 2 year 3 4 4 16 16 16 64 256 4 16 64 16 256 4 4 4 4 4 4 64 64 64 16 64 4 256 256 256 64 16 16 4 4 4 256 64 256 64 16 16 64 16 64

16 256 16 16 4 4 64 64 64 64 4 1024 4 16 64 4 64 64

fractionf ref.g 14 D 23 E 15 A 28 A 28 D 14 F E E E E E F E

F E

14

28 14 14 17 14

14 28 20 17

F

E E E E

18

17 18

D D E E E

17

F E

18

19

21 19

F

17 17


aOdorants

were numbered according to their retention time on the FFAP column. bIdentified by

comparing the retention indices on the FFAP and DB-5 column, the mass spectrum, as well as odor quality and intensity with data obtained from authentic reference standards analyzed in parallel. cOdor quality as perceived during GC-O. dRI = linear retention index. eFD = flavor dilution factor. fSPE fraction in which the odorant was identified; where SPE fractions are not listed, odorants were identified in the unfractionated SAFE isolate. gReference of the compound as a propolis volatile. hMass spectra could not be obtained in the propolis isolates. Identification was based on the remaining criteria as indicated above. iOdorants were not separated on either GC column. jIdentified by P&T-TD-GC-MS.


Page 26 of 34

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Table 4. Concentrations, Odor Thresholds, and Odor Activity Values (OAV) of Odor-Active Compounds in Propolis. no.

odorant

odor quality

45 12 15 48 18 40 27 47 26 17 32 24 16 5 3 23 39 42 20 22 34 19 11 14 37 38

(E)-isoeugenol linalool butanoic acid 3-phenylpropanoic acid 3-methylbutanoic acide eugenol β-ionone vanillin 2-phenylethanol 2-methylbutanoic acide cinnamaldehyde 2-methoxyphenol phenylacetaldehyde 1,8-cineole α-pinene hexanoic acid cinnamyl acetate 2-methoxy-4-vinylphenol 3-methylnonane-2,4-dione (E)-β-damascenone methyl cinnamate (2E,4E)-nona-2,4-dienal (2E)-non-2-enal (2E,6Z)-nona-2,6-dienal γ-decalactone ethyl cinnamate

clove floral sweaty, rancid floral sweaty, rancid clove floral vanilla floral, rose sweaty, rancid cinnamon smoky floral, honey eucalyptus piney rancid cinnamon clove hay cooked apple cinnamon fatty green cucumber coconut cinnamon

aMean

conca (µg/kg) 5180 1300 12600 21600 1850 5790 219 17200 41800 7580 52000 117 2150 965 10200 19300 7830 2570 12.5 44.8 1560 31.1 82.7 2.5 296 735

odor thresholdb (µg/kg) 1.4 3.4 34 79 9.0 30 1.3 140 490 110 790 1.8 34 18 210 460 200 98 0.78 6.2 1300 30 140 65 4800 7100

OAVc 3700 380 750 270 210 190 170 120 85 69 66 65 63 54 49 42 39 26 16 7.2 1.2 1.0

Quantitation and Seasonal Variation of Key Odorants in Propolis

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