Characterization of the Major Odor-Active Compounds in the Rhizome of the Greater Galangal (Alpinia galanga)

Abstract

The rhizomes of Alpinia galanga have a characteristic aroma, the molecular basis of which has not yet been fully clarified. Application of gas chromatography–olfactometry and aroma extract dilution analysis to the volatiles isolated from fresh A. galanga rhizomes by solvent-assisted flavor evaporation led to the detection of 43 odorants with flavor dilution factors of 1–4096. Enantiodifferentiation increased the number to 47. The structures of 36 odorants were elucidated, 18 of which were previously unknown in A. galanga, and 21 were shown to be present in concentrations above the odor threshold concentration (OTC). High odor activity values (OAVs; concentration/OTC) were particularly obtained for 1,8-cineole (130000), (S)-galangal acetate (44000), and myrcene (8000). As proof of success, the characteristic aroma of A. galanga rhizome was reconstructed with 20 major odorants in their natural concentrations. The results will contribute to a deeper molecular understanding of the aroma of Alpinia species and serve as the basis to study aroma changes during processing and culinary use of A. galanga rhizome.

Introduction

Alpinia galanga, also referred to as the greater galangal, is a perennial plant in the ginger family Zingiberaceae. It is cultivated in many Asian countries, particularly in China, India, Indonesia, Malaysia, and Thailand. The used part is the underground rhizome, which has a cylindrical shape and is pale yellow in color. The rhizome is a popular spice in Southeast Asian cuisines. Moreover, it is reported to have antibacterial, antioxidant, antifungal, and antitumor activities. Medical uses owing to its carminative and stomachic properties include the treatment of stomach ache, colic, and diarrhea. The essential oil isolated from A. galanga rhizomes has been applied to flavor beverages and in perfumery.

Fresh A. galanga rhizomes exhibit a eucalyptus leaf-like, fruity, pungent, and floral aroma which substantially differs from that of ginger. Although some work on A. galanga volatiles has been published in the last decades, the information available on the contribution of individual odorants to the overall aroma is limited. Most studies focused on the mere structural identification of volatiles by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) without assessing the odor impact. In summary, A. galanga rhizomes from Malaysia, − Thailand, - India, − and China have been investigated, which led to the identification of almost 100 different volatiles. The most recent study on A. galanga volatiles employed GC×GC–TOFMS and reported a total of 102 compounds among which were 47 hydrocarbons, 25 alcohols, 7 ketones, 7 esters, 3 aldehydes, 4 ethers, and 9 other compounds. Major volatiles include common compounds such as 1,8-cineole, β-caryophyllene, α-terpineol, camphor, terpinen-4-ol, β-bisabolene, α- and β-pinene, camphene, geranyl acetate, and acetic acid, but also some more specific compounds such as β-sesquiphellandrene, 2- and 3-acetoxy-1,8-cineoles, chavicol (4-allylphenol), chavicol acetate, and in particular the (S)-isomer of 1’-acetoxychavicol acetate, i.e., (1S)-1-[4-(acetyloxy)­phenyl]­prop-2-en-1-yl acetate, commonly referred to as (S)-galangal acetate, which is the pungent principle of A. galanga rhizomes. ,

Only in a series of four papers, the odor activity of A. galanga volatiles has been addressed. − The volatiles isolated from fresh rhizomes by a mild vacuum steam-distillation approach were fractionated into a hydrocarbon fraction and a polar compound fraction. The latter exhibited the characteristic aroma and was subsequently subjected to gas chromatography–olfactometry (GC–O) and aroma extract dilution analysis (AEDA), which resulted in ∼20 odor-active compounds. High FD factors were obtained for 1,8-cineole, linalool, geranyl acetate, chavicol acetate, and eugenol followed by bornyl acetate, 2-acetoxy-1,8-cineol (two isomers), methyl eugenol, acetic acid, citronellyl acetate, and (S)-galangal acetate. Despite its only moderately high FD factor, (S)-galangal acetate was considered particularly important because its odor quality was very close to the overall aroma impression of A. galanga rhizomes. Further odor-active volatiles included 4-terpineol (terpinen-4-ol), α-terpineol, thymyl acetate, eugenyl acetate, and chavicol. A closer look into the relative abundances and odor properties of the eight isomeric 2- and 3-acetoxy-1,8-cineol acetates suggested that only the (1R,4S,6R)-isomer of trans-1,3,3-trimethyl-2-oxabicyclo-[2.2.2]­oct-6-yl acetate, i.e. the trans-2-acetoxy-1,8-cineol substantially contributed to the characteristic A. galanga odor.

To achieve a comprehensive understanding of the volatiles contributing to the typical A. galanga rhizome aroma, the aim of our study was to screen the entire volatile fraction isolated from the rhizomes for odor-active compounds by GC–O and AEDA, assign the structures of the most potent odorants, determine their concentrations, calculate their odor activity values (OAVs) as ratio of the natural concentration in the plant material to the odor threshold concentration (OTC) of the compound, and finally reconstitute the aroma to validate the results.

Materials and Methods

Rhizomes

A. galanga rhizomes grown in Thailand were purchased from a local Asian food shop in portions of ∼100 g in 2019–2022. Fresh cuts fully represented the characteristic aroma of A. galanga rhizomes. The rhizomes were frozen with liquid nitrogen and powdered by a SPEX SamplePrep 6875 Freezer Mill (C3 Prozess- und Analysentechnik, Haar, Germany). The powders were stored in brown glass bottles at −24 °C prior to analysis.

Reference Odorants

Compounds 2, 3, 6, 8, 9, racemic 13, 14 (mixture of enantiomers), 14a, 14b, 15, 17a, 17b, 18a, 18b, 21, 25, 26, 29, 32, 36–38, 42, and 43 were purchased from Merck (Darmstadt, Germany), 18 (mixture of enantiomers), 31, and 35 were obtained from TCI (Eschborn, Germany), 27 and 39 were from Fluorochem (Hadfield, UK), 11 and 16 from Thermo Fisher Scientific (Waltham, MA, USA), 24 from Toronto Research Chemicals (Toronto, Canada), and 41 (mixture of enantiomers) was obtained from Biomol (Hamburg, Germany). Odorants 1, 4, and 30 were synthesized as detailed in the literature. Odorant 20 was synthesized from 18b via trans-2-hydroxy-1,8-cineole. , Odorant 13b was obtained from racemic 13 by preparative chiral HPLC as detailed recently. Similarly, 41a and 41b were obtained from the enantiomeric mixture: a solution of 41 in hexane (∼2 mg/mL) was applied to a preparative HPLC system (Knauer, Berlin, Germany) equipped with a MWD 2.1 L detector, a P6.1 L HPG pump, a 6.1 L autosampler, and a Labocol Vario 4000 fraction collector. The column was a chiral Lux Amylose-1S (Phenomenex, Aschaffenburg, Germany). The injection volume was 250 μL. Elution was performed with hexane/methanol (95:5; v:v) at a flow rate of 2.5 mL/min. The collected target peak fractions were dried over anhydrous sodium sulfate and the solvent was finally removed by rotary evaporation resulting in 41a and 41b in 96 and 99% purity (GC–FID). Enantiomeric assignments were based on the odor properties and the enantiomeric distributions previously reported in galangal rhizome. ,,

Stable Isotopically Substituted Odorants

(²H₆)-2 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). (²H₃)-8 was purchased from Merck. (²H₉)-17a was obtained from EQ Laboratories (Augsburg, Germany). (²H₅)-16, (¹³C,²H₂)-β-caryophyllene, (²H₃)-31 and (²H₃)-eugenyl acetate were purchased from aromaLAB (Martinsried, Germany). The following isotopically substituted compounds were synthesized as described in the references provided: (²H₂)-1, (²H₂)-3, (²H₃)-9, (²H_2–3)-13, (²H₆)-18, (²H₂)-15, (²H₃)-21, (²H_2–3)-26, (²H₂)-30, (²H_2–3)-32, (²H₂)-35, (²H₂)-38, (²H₃)-43, (²H₃)-trans-isoeugenol.

Miscellaneous Chemicals and Reagents

Diethyl ether, pentane, and silica gel 60 (0.063–0.2 mm) were obtained from VWR (Darmstadt, Germany). The solvents were freshly distilled before use.

Gas Chromatography

GC–O analyses were carried out by using a gas chromatograph equipped with a cold on-column injector, a flame ionization detector (FID), and a custom-made sniffing port. The column was either a DB-FFAP or an HP-5, both 30 m × 0.32 mm i.d., 0.25 μm film (Agilent Technologies, Waldbronn, Germany). GC–MS analyses were carried out by using a GC–MS system with an ion trap mass spectrometer, a GC×GC–TOFMS system with a Pegasus III time-of-flight (TOF) mass spectrometer (Leco, Mönchengladbach, Germany), or a heart-cut GC–GC–HRMS system with an Orbitrap mass spectrometer (Thermo Fisher Scientific). Further details on the GC systems are available in the Supporting Information file.

AEDA

Diethyl ether (200 mL) and sodium sulfate (60 g) were added to rhizome powder (20 g). The mixture was stirred at room temperature overnight and then filtered. The filtrate was subjected to solvent-assisted flavor evaporation (SAFE). SAFE was performed at 40 °C over a period of 30 min. The volatile isolate was concentrated to 1 mL by using a Vigreux column (50 cm × 1 cm) and a microdistillation device at a water bath temperature of 40 °C. The concentrated volatile isolate (1 mL) was stepwise diluted 1:2 with diethyl ether to obtain dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, and 1:4096. The undiluted sample, as well as each diluted sample, was analyzed by GC–O using the FFAP column. Each odorant was characterized by its retention index, its odor as perceived at the sniffing port, and its FD factor. The retention index was calculated from the retention time of the odorant and the retention times of adjacent n-alkanes by linear interpolation. The FD factor was determined as the dilution factor of the highest diluted sample in which the odorant was detected at the sniffing port during GC–O by any of three trained assessors. An FD factor of 1 was assigned to odorants that were only detected in the undiluted sample.

Preliminary structure assignments were achieved by comparing RIs and odor properties of the odorants in the A. galanga volatile isolate to data compiled in the VCF database, the Leibniz-LSB@TUM Odorant Database, and previous literature on A. galanga volatiles. Structure proposals were confirmed by GC–O analyses of authentic reference odorants, which were performed in parallel to the analysis of the A. galanga volatile isolate, using the FFAP and the HP-5 column. Final structure confirmation was achieved by mass spectrometry in EI and CI modes. To reduce coelution problems, the volatile isolate was fractionated by using liquid chromatography. In detail, pentane (2 mL) was added to a concentrated A. galanga volatile isolate and the mixture was reconcentrated to 0.5 mL to remove the extraction solvent diethyl ether. The pentane solution was applied onto a slurry of purified silica gel (8 g) in pentane inside a water-cooled glass column (12 °C; 1 cm i.d.). Elution was performed with pentane/diethyl ether mixtures in ratios of 100:0, 90:10, 70:30, 50:50, and 0:100 (v:v; 50 mL each). The eluate was collected in 50 mL portions. Each fraction was dried over anhydrous sodium sulfate and concentrated to 0.5 mL. The A. galanga odorants were localized in the fractions by GC–O before the fractions were subjected to GC–MS, GC×GC–TOFMS, or heart-cut GC–GC–HRMS analysis.

Quantitation Assays

For GC–MS quantitations, rhizome powder (1–50 g) was spiked with stable isotopically substituted internal standards (∼0.07 to 145 μg), diethyl ether (50–500 mL) was added, and the mixture was stirred at room temperature overnight. After filtration and drying over anhydrous sodium sulfate, nonvolatiles were removed by SAFE. The volatile isolates were appropriately concentrated and analyzed in CI mode using the one-dimensional GC–MS system (2, 8, 13, 14, 15, 18, 27, 31, 35, 36, 39, and 43) or the two-dimensional heart-cut GC–GC–HRMS with the column combinations DB-FFAP/DB-1701 (1, 9, 16, 17a, 17b, 21, 24, 26, 30, 32) or DB-FFAP/BGB-176 (20, 38). Peak areas associated with the analyte and internal standard were obtained from the extracted ion chromatograms using specific quantifier ions. Odorant concentrations in the A. galanga rhizome were finally calculated from the area counts of the analyte peak, the area counts of the standard peak, the amount of standard added, and the amount of rhizome used by employing a calibration line equation obtained from the analysis of analyte/standard mixtures in five different concentration ratios (∼1:5, 1:2, 1:1, 2:1, 5:1) and subsequent linear regression. Individual quantifier ions and calibration line equations are provided in the Supporting Information file (Table S1).

For the GC–FID quantitations of compounds 3 and 41 after exhaustive extraction, a water-cooled glass column (12 °C, 1 cm i.d.) was successively filled with a plug of defatted cotton wool, a layer of sea sand (1 cm), a layer of A. galanga rhizome powder (∼1 g), and finally another layer of sea sand (1 cm). Continuous extraction was performed at ∼2 mL/min with diethyl ether (1 L) and the eluate was collected in five 200 mL portions. Tridecane (568 μg) was added to each portion as internal standard. The portions were concentrated (20 mL) using a Vigreux column (50 cm × 1 cm) and the concentrates were directly subjected to GC–FID analysis without further purification. The concentrations of 3 and 41 were finally calculated from the area counts of the corresponding peaks, the area counts obtained for the internal standard tridecane, the exact amount of A. galanga rhizome powder used, and response factors determined from the parallel analysis of a ∼1:1:1 reference compound mixture of 3, 41, and tridecane.

Determination of Odor Threshold Concentrations

OTCs in water were determined orthonasally by employing the standard procedure of the American Society for Testing and Materials (ASTM). Odorant concentrations increased by a factor of 3 between consecutive three-alternative forced choice tests. Samples were provided in covered cylindrical PTFE vessels (5.7 cm height × 3.5 cm i.d.). The tests were performed by 15–20 nonsmoking, healthy, and trained assessors, 5–6 males and 9–11 females, aged 21–60, in a special sensory room with separated booths and controlled temperature (22 ± 2 °C).

Preparation of the Aroma Reconstitution Model

First, individual aqueous or ethanolic stock solutions of the reference odorants were prepared. These were sourced as detailed before except for 41, which was isolated from a hexane/acetone (10:1; v:v) extract (1.5 L) obtained from fresh A. galanga rhizome (100 g) by the preparative HPLC approach detailed in the Reference Odorants section. Aliquots of the stock solutions were then combined and diluted with water to generate a mixture with odorant concentrations identical to the natural concentrations previously determined in the A. galanga rhizome. The final concentration of ethanol in the aroma reconstitution model was kept below its OTC in water (1 g/L). A small amount (10 g/kg) of low odor sunflower oil was finally added to mimick the natural lipid content of A. galanga rhizome.

Quantitative Olfactory Profile Analysis

Fresh cuts of galangal rhizomes (5 g) and aroma reconstitution model in covered cylindrical ground neck glasses (5.7 cm height, 3.5 cm i.d.) were simultaneously presented to 16 nonsmoking, healthy, and trained assessors (10 females, 6 males; aged 21–60). The assessors were asked to rate the intensities of six predefined descriptors on a scale from 0 to 3 with 0.5 increments (0 = not detectable, 1 = weak, 2 = moderate, and 3 = strong). Each descriptor was defined by the odor of an aqueous solution of a reference compound in a concentration of ∼ 100 times above its OTC. The six odor descriptors and the respective reference compounds were “eucalyptus leaf-like” (1,8-cineole), “fruity, pungent” (galangal acetate), “floral, citrusy” (linalool), “green, grassy” ((3Z)-hex-3-enal), “clove-like” (eugenol), “geranium leaf-like” (myrcene). The assessors were also asked to rate the similarity of the model to the A. galanga rhizome in the overall olfactory profile on a scale from 0 to 3 with 0.1 increments (0 = no similarity, 1 = weak similarity, 2 = clear similarity, and 3 = identical). Ratings of the assessors were averaged by calculating the arithmetic mean.

Results and Discussion

Screening for A. galanga Rhizome Odorants

Application of GC–O on the volatiles isolated from the fresh rhizome by solvent extraction and SAFE resulted in the detection of 43 odorous regions in the chromatogram (Table ). AEDA revealed FD factors between 1 and 4096. Structures could be assigned to 33 odorous regions, which included the 10 with the highest FD factors (128–4096). Eighteen of the identified odorants were previously unknown in A. galanga rhizome.

1. Odorants in the Volatile Isolate Obtained from A. galanga Rhizome.

no.	odorant(s)	odor	RI FFAP	RI HP-5	FD factor	previously reported
1	(3Z)-hex-3-enal	green, grassy	1137	802	64
2	myrcene	geranium leaf	1156	987	64	4,6,7,11–19
3	1,8-cineole	eucalyptus leaf	1193	1024	4096	4,6,7,11–21
4	2-acetyl-1-pyrroline	popcorn	1323	926	2
5	unknown	eucalyptus leaf	1373		4
6	heptyl acetate	fruity	1380	1115	1
7	unknown	earthy	1419		64
8	acetic acid	vinegar	1435	<700	256	7
9	methional	cooked potato	1442	904	128
10	unknown	moldy	1455		2
11	octyl acetate	fruity	1467	1212	1
12	unknown	fatty	1516		16
13	linalool	floral, citrusy	1537	1095	32	4,6,7,11,13,15–21
14	terpinen-4-ol	moldy	1588	1183	64	4,6,7,12–17,19,21
15	butanoic acid	cheesy, sweaty	1615	810	4
16	phenylactaldehyde	floral, honey	1630	1044	32
17	2-/3-methylbutanoic acid	cheesy, sweaty	1663	880	32
18	α-terpineol	citrusy, floral	1690	1175	8	4,6,7,11–21
19	unknown	moldy	1711	1514	4
20	trans-2-acetoxy-1,8-cineole	eucalyptus leaf	1716	1348	128	7–11
21	pentanoic acid	cheesy, sweaty	1733	909	128
22	unknown	citrusy	1753		64
23	unknown	moldy	1758		64
24	germacrene B	mushroom	1805	1571	32
25	p-cymen-8-ol	citrusy	1837	1185	4	11,13,16,19,21
26	γ-octalactone	coconut	1903	1260	64
27	chavicol acetate	mushroom	1941	1352	256	4,7,14,16,19
28	unknown	eucalyptus leaf	1958		64
29	caryophyllene oxide	citrusy	1959	1570	4	4,13,15–19,21
30	trans-4,5-epoxy-(2E)-dec-2-enal	metallic	1991	1375	512
31	methyl eugenol	clove	2006	1405	16	6,7,16–19
32	γ-nonalactone	coconut	2012	1369	16
33	unknown	metallic	2052		8
34	unknown	clove	2100		32
35	cinnamyl acetate	fruity	2121	1450	64
36	eugenol	clove	2155	1356	4096	7,9,12,13,16–19
37	sotolon	fenugreek	2188	1110	4
38	4-propylphenol	phenolic	2260	1258	64
39	chavicol	phenolic	2333	1258	32	4,7,11,16,18,19
40	unknown	fatty, green	2384	1688	16
41	galangal acetate	fruity, pungent	2480	1648	4096	3,7,9,22
42	phenylacetic acid	honey, beeswax	2543	1270	2
43	vanillin	vanilla	2557	1407	128

^aEach odorant was identified by comparing its retention indices on two GC columns of different polarity (DB-FFAP, HP-5), its mass spectrum obtained by GC–MS or GC×GC–MS, and its odor quality as perceived at the sniffing port during GC–O analysis to the respective data of authentic reference compounds analyzed under the same conditions.

^bOdor quality as perceived at the sniffing port during GC–O analysis.

^cRetention index; calculated from the retention time of the compound and the retention times of adjacent n-alkanes by linear interpolation.

^dFlavor dilution factor; dilution factor of the highest diluted sample of the A. galanga volatile isolate in which the odorant was detected during GC–O analysis by any of three experienced sniffers.

^eReference(s) reporting the compound as A. galanga volatile; no entry means that compounds have not yet been reported.

^fAn unequivocal mass spectrum of the compound could not be obtained from the analysis of the A. galanga volatile isolate, identification was based on the remaining criteria detailed in footnote a.

^g2-methylbutanoic acid (17a) and 3-methylbutanoic acid (17b) were not separated on the GC column used for AEDA; however, the EI mass spectrum indicated a mixture of both isomers.

The highest FD factor of 4096 was determined for eucalyptus leaf-like smelling 1,8-cineole (eucalyptol; 3), clove-like smelling eugenol (36), and galangal acetate (41), which was described as fruity and pungent. High FD factors were also obtained for trans-4,5-expoxy-(2E)-dec-2-enal (30; metallic; FD factor 512), acetic acid (8; vinegar-like; FD factor 256), chavicol acetate (27; mushroom-like; FD factor 256), methional (9; cooked potato-like; FD factor 128), trans-2-acetoxy-1,8-cineole (20; eucalyptus leaf-like, FD factor 128), pentanoic acid (21; cheesy, sweaty; FD factor 128), and vanillin (43; vanilla-like; FD factor 128). Another 23 odorants showed FD factors between 1 and 64.

The three most potent odorants in the A. galanga volatile isolate, namely 1,8-cineole (3), eugenol (36), and galangal acetate (41) have all previously been reported as A. galanga volatiles ,,,- , and also as important odor-active compounds. , Further compounds already known as A. galanga odorants included, acetic acid (8), linalool (13), terpinen-4-ol (14), α-terpineol (18), trans-2-acetoxy-1,8-cineole (20), chavicol acetate (27), methyl eugenol (31), and chavicol (39). By contrast, we did not confirm odor-active amounts of bornyl acetate, citronellyl acetate, geranyl acetate, thymyl acetate, eugenyl acetate, and thymol. , On the other hand, (3Z)-hex-3-enal (1), 2-acetyl-1-pyrroline (4), heptyl acetate (6), methional (9), octyl acetate (11), butanoic acid (15), phenylactaldehyde (16), 2-methylbutanoic acid (17a), 3-methylbutanoic acid (17b), pentanoic acid (21), germacrene B (24), γ-octalactone (26), trans-4,5-epoxy-(2E)-dec-2-enal (30), γ-nonalactone (32), cinnamyl acetate (35), sotolon (37), 4-propylphenol (38), phenylacetic acid (42), and vanillin (43) have not been reported as A. galanga volatiles before. Myrcene (2), p-cymen-8-ol (25), and caryophyllene oxide (29) are well-known A. galanga volatiles, but have not been identified as odor-active compounds before.

Enantiomeric Distribution of Chiral Odorants

The odorants detected in the AEDA (cf. Table ) included chiral compounds such as linalool (13), terpinen-4-ol (14), α-terpineol (18), and galangal acetate (41). Individual enantiomers of chiral odorants may substantially differ in their odor properties. For example, (R)- and (S)-linalool are quite similar in their odor quality, the (R)-isomer, however, is the more potent odorant. Recently it has been shown that the OTCs of (R)- and (S)-linalool in water differ by a factor of 10. Galangal acetate, which due to its high FD factor in the AEDA is of particular interest, is another example. (S)-galangal acetate was reported to show a strong pungent, fruity, and ginger-like odor, whereas the odor of the (R)-isomer was described as weak, faint woody, and sweet. ,

To be able to correctly assess the odor contribution of the chiral compounds when calculating OAVs and to consider the exact concentrations of the optical isomers in the aroma reconstitution experiment, we determined the enantiomeric distribution of linalool, terpinen-4-ol, α-terpineol, and galangal acetate. This was achieved by GC–GC–HRMS with heart-cutting and a chiral column in the second dimension.

The results (Table ) showed that the linalool in the A. galanga rhizome was pure (S)-linalool; no (R)-linalool was detected. By contrast, both enantiomers were present in substantial amounts in terpinen-4-ol and α-terpineol, with R/S ratios of 62/38 and 16/84, respectively. In galangal acetate, the (S)-enantiomer clearly dominated with 96%. This result was consistent with earlier data of Yang et al., who reported a R/S ratio of the galangal acetate in A. galanga rhizomes of approximately 5/95. To the best of our knowledge, no data on the enantiomeric distribution of linalool, terpinen-4-ol, and α-terpineol in A. galanga rhizome have been published to date.

2. Enantiomeric Distribution of Important Chiral Odorants in A. galanga Rhizome.

no.	odorant	ratio R/S (%)
13	linalool	0/100
14	terpinen-4-ol	62/38
18	α-terpineol	16/84
41	galangal acetate	4/96

Odorant Concentrations and OAVs

Major odor-active compounds resulting from the odorant screening by GC–O and AEDA were subsequently quantitated in the A. galanga rhizome. The majority of odorants were quantitated by GC–MS using isotopically substituted odorants as internal standards, preferentially isotopologues of the target compounds (cf. Supporting Information file, Table S1). Due to their enormously high concentrations, this was not possible for 1,8-cineole and galangal acetate. An economically viable quantity of the isotopologue corresponded to a rhizome sample amount far below 1 g, which was considered unsuitable for ensuring representativeness and accuracy. Therefore, 1,8-cineole and galangal acetate were quantitated by GC–FID after exhaustive solvent extraction (cf. Supporting Information file, Table S2). The individual concentrations of the linalool, terpinen-4-ol, and α-terpineol enantiomers were calculated from the sum of enantiomers as determined by GC–MS or GC–FID and the enantiomeric ratio provided in Table .

The results of the quantitations (Table , column 3) revealed odorant concentrations ranging from 2.25 μg/kg for γ-nonalactone (32) to 4210 mg/kg for (S)-galangal acetate (41b), thus covering more than six powers of ten. In addition to (S)-galangal acetate, particularly high concentrations were also obtained for 1,8-cineole (3; 530,000 μg/kg), acetic acid (8; 360,000 μg/kg), (R)-galangal acetate (41a; 133,000 μg/kg), chavicol acetate (27; 127,000 μg/kg), germacrene B (24; 62,700 μg/kg), and chavicol (39; 31,100 μg/kg). To the best of our knowledge, this is the first comprehensive report of A. galanga odorant concentration data.

3. Concentrations and OAVs of Major Odorants in A. galanga Rhizome.

no.	odorant	concentration (μg/kg)	OTC (μg/kg)	OAV
3	1,8-cineole	530,000	4.0	130000
41b	(S)-galangal acetate	4,210,000	96	44000
2	myrcene	9630	1.2	8000
24	germacrene B	62,700	27	2300
36	eugenol	2950	1.8	1600
27	chavicol acetate	127,000	130	980
13b	(S)-linalool	7050	8.3	850
1	(3Z)-hex-3-enal	72.8	0.12	610
39	chavicol	31,100	140	220
8	acetic acid	360,000	5600	64
20	trans-2-acetoxy-1,8-cineole	8760	230	38
31	methyleugenol	8180	240	34
14a	(R)-terpinen-4-ol	2360	110	21
30	trans-4,5-epoxy-(2E)-dec-2-enal	2.37	0.22	11
9	methional	3.36	0.43	7.8
41a	(R)-galangal acetate	133,000	23,000	5.8
16	phenylactaldehyde	27.0	5.2	5.2
18b	(S)-α-terpineol	9790	2300	4.3
35	cinnamyl acetate	479	150	3.2
14b	(S)-terpinen-4-ol	1430	590	2.3
43	vanillin	112	53	2.1
26	γ-octalactone	3.47	6.5	<1
38	4-propylphenol	36.6	150	<1
32	γ-nonalactone	2.25	9.7	<1
18a	(R)-α-terpineol	1930	18,000	<1
15	butanoic acid	53.3	2400	<1
17b	3-methylbutanoic acid	3.09	490	<1
17a	2-methylbutanoic acid	4.42	3100	<1
21	pentanoic acid	13.6	11,000	<1

^aMean of triplicates; individual data and standard deviations are available in the Supporting Information file, Table S3.

^bOrthonasal odor threshold concentration in water; OTCs in water of 14 (mixture of enantiomers), 18 (mixture of enantiomers), 20 ((1R,4S,6R)-1,3,3-trimethyl-2-oxabicyclo­[2.2.2]­octan-6-yl acetate), 24, 27, 31, 39 and racemic 41 were determined within this study according to the ASTM E679–19 standard practice; OTCs in water of 14a, 14b, 18a, 18b, 41a, and 41b were approximated from their OTCs in air and the OTC of the enantiomeric mixture in water (cf. Supporting Information file, Table S4; the remaining OTCs were taken from the Leibniz-LSB@TUM Odorant Database.

^cOdor activity value; calculated as the ratio of the concentration in the A. galanga rhizome to the OTC of the respective odorant in water.

Moreover, the concentration data were the basis to better assess the odor potency of the individual A. galanga odorants by calculating OAVs. Among the 29 odorants quantitated, 21 showed an OAV >1 (Table , rightmost column; Figure ). The highest OAV of 130000 was calculated for eucalyptus leaf-like smelling 1,8-cineole (3). This was consistent with the high FD factor of 4096 determined for this compound in the AEDA. High OAVs were also obtained for fruity, pungent smelling (S)-galangal acetate (41b; OAV 44000), geranium leaf-like smelling myrcene (2; OAV 8000), mushroom-like smelling germacrene B (24; OAV 2300), clove-like smelling eugenol (36; OAV 1600), mushroom-like smelling chavicol acetate (27; OAV 980), floral, citrusy smelling (S)-linalool (13b; OAV 850), green, grassy smelling (3Z)-hex-3-enal (1; OAV 610), and phenolic smelling chavicol (39; OAV 220). Further 11 odorants showed OAVs between 2.1 and 64. In contrast, the OAVs of eight odorants were <1, i.e., their concentrations in the A. galanga rhizome were below their odor threshold concentrations. Thus, these compounds were considered not to substantially contribute to the overall aroma of A. galanga rhizome and omitted in the subsequent aroma reconstitution experiment.

1.

Structures of major A. galanga odorants together with their odor characteristics and OAVs.

Aroma Reconstitution

To validate the screening and quantitation data and to ensure that no important odorant has been overlooked, successful aroma reconstitution is essential. , Thus, we aimed at preparing a model solution containing the A. galanga rhizome odorants for which OAVs >1 had been determined in their natural concentrations (cf. Table ) and in an appropriate matrix. We were finally successful to include 20 of the 21 odorants with OAVs >1; only germacrene B had to be omitted due to its limited commercial availability. Corresponding to the high moisture content of fresh galangal rhizomes, which amounts to ∼90%, water was selected as the basic matrix component. A minor amount of oil was added to mimick the natural lipid content of the rhizomes, which may substantially impact the release behavior of the odorants. The aroma reconstitution model was then subjected to a quantitative olfactory profile analysis. Trained assessors rated six attributes characterizing typical aroma notes of A. galanga rhizome. Fresh cuts of galangal rhizomes were evaluated in parallel as a reference.

Results revealed an excellent agreement between the olfactory profile of the reconstitution model and that of the A. galanga rhizome (Figure ). The overall similarity was rated 2.7 out of 3. Both profiles showed a dominating eucalyptus leaf-like aroma note, a substantial fruity, pungent note and somewhat weaker floral/citrusy, clove-like, green/grassy, and geranium leaf-like aspects.

2.

Quantitative olfactory profiles of the aroma reconstitution model solution (20 compounds with OAVs >1) in comparison to the quantitative olfactory profile of the A. galanga rhizome. Assessors rated the intensity of each descriptor on a scale from 0 to 3 in 0.5 increments with 0 = not detectable, 1 = weak, 2 = moderate, and 3 = strong.

The aroma reconstitution results provided evidence that the combination of mild, artifact-avoiding volatile isolation, odorant screening by GC–O and AEDA, thorough structure assignment including enantiodifferentiation, and exact quantitation with the help of isotopically substituted internal standards, was successful in deciphering the major odor-active compounds in A. galanga rhizome. The results provide the foundation for further investigations on the aroma differences between different Alpinia species and other edible members of the ginger family and studies on the molecular background of aroma changes during processing and culinary use of A. galanga rhizomes.

Glossary

Abbreviations

AEDA

aroma extract dilution analysis

CI

chemical ionization

EI

electron ionization

FD

flavor dilution

FFAP

free fatty acid phase

GC–O

gas chromatography–olfactometry

GC×GC

comprehensive two-dimensional gas chromatography

HRMS

high-resolution mass spectrometry

OAV

odor activity value

OTC

odor threshold concentration

SAFE

solvent-assisted flavor evaporation

TOFMS

time-of-flight mass spectrometry.

Glossary

Nomenclature

trans-2-Acetoxy-1,8-cineole

trans-1,3,3-trimethyl-2-oxabicyclo­[2.2.2]­octan-6-yl acetate

2-acetyl-1-pyrroline

1-(3,4-dihydro-2H-pyrrol-5-yl)­ethan-1-one

caryophyllene oxide

(1R,4R,6R,10S)-4,12,12-trimethyl-9-methylidene-5-oxatricyclo­[8.2.0.0^4,6]­dodecane

chavicol

(4-prop-2-en-1-yl)­phenol

chavicol acetate

(4-prop-2-en-1-yl)­phenyl acetate

1,8-cineole (eucalyptol)

1,3,3-trimethyl-2-oxabicyclo­[2.2.2]­octane

cinnamyl acetate

(2E)-3-phenylprop-2-en-1-yl acetate

p-cymen-8-ol

2-(4-methylphenyl)­propan-2-ol

trans-4,5-epoxy-(2E)-dec-2-enal

trans-(2E)-3-(3-pentyloxiran-2-yl)­prop-2-enal

eugenol

2-methoxy-4-(prop-2-en-1-yl)­phenol

galangal acetate

1-[4-(acetyloxy)­phenyl]­prop-2-en-1-yl acetate

germacrene B

(1E,5E)-1,5-dimethyl-8-(propan-2-ylidene)­cyclodeca-1,5-diene

linalool

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

methional

3-(methylsulfanyl)­propanal

methyleugenol

1,2-dimethoxy-4-(prop-2-en-1-yl)­benzene

myrcene

7-methyl-3-methylideneocta-1,6-diene

γ-nonalactone

5-pentyldihydrofuran-2­(3H)-one

γ-octalactone

5-butyldihydrofuran-2­(3H)-one

sotolon

3-hydroxy-4,5-dimethylfuran-2­(5H)-one

terpinene-4-ol

4-methyl-1-(propan-2-yl)­cyclohex-3-en-1-ol

α-terpineol

2-(4-methylcyclohex-3-en-1-yl)­propan-2-ol

vanillin

4-hydroxy-3-methoxybenzaldehyde

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.5c02750.

• Detailed information on the GC systems used; internal standards, quantifier ions, and calibration lines used in the GC–MS quantitations; primary results obtained from the exhaustive extraction approach applied to the quantitation of 1,8-cineole and galangal acetate; individual odorant concentrations used for mean calculations and standard deviations; and approximation of enantiospecific OTCs in water for odorants 14, 18, and 41 (PDF)

J.S. gratefully acknowledges funding from the China Scholarship Council (CSC), grant no. 201806300033.

The authors declare no competing financial interest.