Published online 30 March 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ffj.1633
Analysis of enantiomeric linalool ratio in green and roasted coffee
Bernd Bonnländer,1* Roberto Cappuccio,1 Furio Suggi Liverani1 and Peter Winterhalter2
1 Illycaffè S.p.a., Via Flavia 110, 34147 Trieste, Italy
2 Institute of Food Chemistry, Technical University of Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany Received 24 September 2004; Revised 7 April 2005; Accepted 19 April 2005
ABSTRACT: The enantiomeric distribution of the monoterpene alcohol linalool 3,7-dimethylocta-1,6-dien-3-ol was determined by multi-dimensional gas chromatography (MDGC) and dual-column switching (DCS) GC–MS for the first time in green and roasted coffee, after extraction by simultaneous distillation–extraction (SDE) and stir-bar sorptive extraction (SBSE). The sensory impact of the two enantiomers was evaluated in water and by addition experiments to espresso beverage. Post-harvest treatment of coffee seems to shift the enantiomeric ratio from racemic in washed coffee to an excess of S-(+)-linalool in dry processed coffee. Copyright © 2006 John Wiley & Sons, Ltd.
KEY WORDS: enantiomeric composition; linalool; coffee; espresso; multi-dimensional gas chromatography (MDGC); dual column switching (DCS); stir-bar sorptive extraction (SBSE)
* Correspondence to: B. Bonnländer, Aromalab–Illycaffè, Padriciano 99, 34012 Trieste, Italy.
E-mail: [email protected]
Introduction
Linalool (3,7-dimetyl-1,6-octadien-3-ol) is an important flavour and fragrance compound. It contributes to the characteristic aroma of a vast number of natural products, such as fruits and spices, as well as tea and chocolate.1–4 Two optical isomers with different odour profiles and thresholds are described: 3S-(+)-linalool is perceived as sweet, floral, petitgrain-like (odour threshold 7.4 ppb) and the 3R-form as more woody and lavender-like (odour threshold 0.8 ppb).5–6
Plants mostly produce only one linalool isomer, so that the enantiomeric excess can be used as an indicator for authenticity. The R-form, for example, prevails in basil oil (Ocimum basilicum L.) and Japanese pepper (Xanthoxylum piperitum DC.), whereas the 3S-(+)-linalool dominates in orange oil, strawberries and cocoa products.7 Plants with a balanced distribution of both enantiomers are also known (e.g. pineapple).1 Recently, changes in the enantiomeric composition during beer production have been monitored using stable isotope dilution analysis.8
In coffee, enantiomeric separation of chiral compounds has been reported only for short-chain Strecker prod- ucts,9–10 aliphatic alcohols and esters, as well as limonene, α-terpineol and 2-methyltetrahydrofuran-3-one.9 There has been speculation about the influence of coffee pro- cessing and beverage preparation on the chirality of small molecules.9
This paper establishes the enantiomeric composition of linalool in green and roasted Arabica coffee and coffee
beverages, using simultaneous distillation–extraction (SDE) as well as the gentle isolation technique of stir-bar sorptive extraction (SBSE) in combination with MDGC and MDGC–MS. The sensory impact of the addition of linalool enantiomers to espresso coffee and the respective recognition thresholds was evaluated by an experienced coffee-tasting panel.
Materials and Methods Coffee
Green coffee beans (Coffea arabica) originating from the Dominican Republic, Brazil and Guatemala were ground after liquid nitrogen freezing. All single-origin coffee was freshly roasted in a laboratory-scale prototype roaster (Research and Technical Development, Illycaffè). Colour was measured by a prototype laboratory scale leucometer to a colour value of 38 (LKB, Dr Lange, Berlin, Ger- many). Grinding of roast coffee was performed using a conical disk grinder (Super Jolly, Mazzer Luigi, Venice, Italy). The preparation of espresso beverage was achieved using a Gaggia home-user espresso machine under stand- ard conditions.11 Dry post-harvest treatment is character- ized by drying of the whole cherry (bean, mucilage and pulp) on patios or racks in the sun or by mechanical dry- ers. In the washed or wet process, the harvest is passed through washer separators (floaters). The ripe cherries are pulped and the removal of the mucilage is archieved either mechanically or by fermentation. To assure identi- cal sample material, the dry processed sample was hand- selected to eliminate over-ripe or unripe cherries, as eliminated during floating and pulping in the wet process.
Extraction Techniques
The green and roast coffee volatiles were extracted by SDE (Normschliff Gerätebau, Wertheim, Germany) from ground coffee powder. In coffee beverages, SBSE (Twi- ster, Gerstel, Muehlheim, Germany) was used to extract the aroma compounds directly from the cup. A compari- son between SDE12 and SPME, a technique similar to, but less sensitive than, SBSE, has been recently published.13
Instrumental Analysis
The MDGC system consisted of two Fisons Mega 8000 gas chromatographs coupled by a Moving Column Stream Switching13 (MCSS; Carlo Erba, Germany) de- vice. The pre-column was a Carbowax (30 m × 0.25 mm, 0.25 df; ZB-Wax Phenomenex, Torrance, CA, USA), ramped from 35 °C at 4 °C/min to 240 °C. Split injection was applied, with a constant head pressure of 235 kPa helium. Pressure in the dome was set to 125 kPa for the Lipodex [heptakis (2,3,6-tri-O-methyl)-β-cyclodextrine/
polysiloxane; M&N, Germany] main column. Both detec- tors were flame ionization detectors (FIDs).
The MDGC–MS was built in a 5973N GC–MS system (Agilent, Waldbronn, Germany). A Carbowax pre-column (60 m × 0.25 mm; ZB-Wax Phenomenex, Torrance, CA, USA) was coupled via dual-column switching (DCS, Gerstel) device to the same main column as in MDGC.
Liquid extracts were injected in split mode (1:5), Twister (length 1 cm, film thickness 0.5 mm) was desorbed in TDSA (thermal desorption unit and autosampler; TDS2, Gerstel) in splitless mode and trapped in a programmable temperature vapourizer (PTV), which was in solvent venting mode at 50 ml helium flow during desorption, rapidly ramped from −120 °C to 250 °C and starting run at constant pressure of 195 kPa. Pressure in the cross piece was kept at 46 kPa during a ramp from 70 °C to 110 °C at 5 °C/min on the pre- and main column, followed by an isothermal period at 90 °C and heat out.
Control flow was switched off to transfer the linalool peak from 34.8 to 35.5 min to the chiral column, analys- ing by SIM in electron impact for ions, m/z 71, 93, 121 and 55 (100 ms dwell time each).
Sensory Analysis
Threshold determination
The recognition thresholds can be defined as ‘the level of the stimulus at which the stimulus can be recognized’17 and it is higher than the detection threshold, i.e. the lowest stimulus which gives a sensation. The detection threshold of linalool in air and water have been reported,6 but no determination in coffee is available in the literature.
We determined the recognition threshold of both R- and S-linalool in water and espresso coffee. It must be pointed out that sensory thresholds are not reproducible and vary over an extremely wide range, especially when a complex system like linalool in coffee is taken into consideration.
The three-alternative forced choice (3-AFC) method of sample presentation was adopted to determine the thresh- olds. Three samples at a time were presented: two con- trols (MQ water) and one sample with linalool in MQ water, asking which one was odorous. The samples were presented with three-digit-coded glasses to a panel of six assessors, trained in coffee tasting, with increasing con- centrations of 0.3 th, 1 th, 3 th and 6 th, where th is a tentative threshold. The same experimental procedure was applied to the threshold determination of the linalool enantiomers in espresso beverage prepared from the same commercial blend. The panel consisted of four females and two males (aged 24 – 45).
The best estimate threshold (BET) for each assessor is the geometric mean of the highest concentration missed and the next higher concentration. The group BET is the geometric mean of the individual BETs.17
Descriptive Analysis
The same panel evaluated five samples of espresso, from the same commercial blend, with medium roasting de- gree. In four out of five samples, R- or S-linalool was added in two different concentrations, two and six times above the threshold level of each isomer (Table 2).
Samples were served monadically in a randomized order, to eliminate presentation effects. Each panellist received 20 ml coffee in a three-digit-coded plastic cup and evaluated it, giving marks on a discrete nine-point scale with semantic anchors (1, barely perceivable–9, extremely intense) to a subset of characteristics drawn out from a set of attributes for taste and aroma commonly used in coffee evaluation. Data was treated with analysis of variance (ANOVA) to detect possible significant dif- ferences in the sensory descriptors.
Reference Samples
Samples of 3R- and 3S-linalool were a gift from Dr C.
Margot, Firmenich S.A. (Basel, Switzerland); 3R- and racemic linalool was commercially available from Aldrich (Milan, Italy).
Results and Discussion
In MDGC, a switching device is used to transfer the target compounds after pre-separation on a first column onto a second column with different separation
characteristics. In the present case, the first separation of the SDE extract of green coffee was performed on a polar Carbowax column. A permethylated cyclodextrine phase was used as second column in order to achieve separation of the enantiomers of linalool. By using only FID detection, one has to be aware that even with a very small cut window, more compounds than only the linalool peak are possibly transferred from the complex mixture of coffee volatiles using the MCSS system. For clear and undoubtful identification of the isomers eluting from the chiral column, mass spectrometry had to be applied, even though the MDGC system already sug- gested enantiomeric excess of S-(+)-linalool. The MS identification is especially crucial in roasted coffee, as the number of known volatile compounds rises from 300 for green coffee to about 1000 in roasted coffee. A schematic outline of the system used for the analysis is shown in Figure 1; the resulting chromatogram for an espresso from Guatemala coffee is presented in Figure 2. The analysis confirmed a small enantiomeric excess of the S- enantiomer of linalool (ee 13.2% S). SBSE was used to extract linalool from coffee preparations in a gentler manner, as racemization of linalool under acidic extrac- tion conditions is known to occur.14 However, no differ- ences in the enantiomeric ratio were observed between
Table 1. Enantiomeric excess in Arabica coffees from different origins and post-harvest treatments
Origin (G/RG) Post-harvest % ee (S)-Linalool (± SD, n = 6)
Brazile RG Dry 22.8 ± 1.7
Guatemala Washed 8.9 ± 1.6
Santa Domingo Dry 45.0
Santa Domingo Washed 11.7
G, raw coffee; RG, roast and ground.
the extraction procedures. Nevertheless, SBSE seems to be more favourable, since the total time for the analysis could be dramatically reduced, as extraction took only 20 min at room temperature.
Analyses of coffees from different origins revealed massive differences (see Table 1) in the enantiomeric ratios of linalool, thus indicating that linalool can not in this case be used as an indicator of authenticity. After examination of the sample’s history and analysis of identical coffee samples from Santo Domingo processed by the dry and the wet procedure, the observed differ- ences could be attributed to the post-harvest method (see Table 1). A possible explanation for the change of enantiomeric excess in favour of S-(+)-linalool for dry
Figure 1.
Table 2. ANOVA, with post hoc Tukey test at 5% of the five samples from a com- mercial blend espresso (ESP) and addition of R- and S-linalool
Analysis ESP LOW-R HIGH-R LOW-S HIGH-S Comp. F Probability
Sweet 4.5 5.33 4.8 4.25 3.25 7.82 0.0006
A A A AB B ***
Caramel 2.83 2.6 5.33 4 0 23.09 < 0.0001
B B A AB C ***
Honey 0 2.25 4.33 0 0 100.38 < 0.0001
C B A C C ***
Flowery 0 0 4.4 4.67 7 32.8 < 0.0001
C C B B A ***
Woody 0 0 0 5 0 53.57 < 0.0001
B B B A B ***
*** Significant at 0.1%; same letters refer to discriminated groups.
HIGH, six times tentative threshold; LOW, twice tentative threshold.
Figure 2.
processed coffee could be due to the aroma precursors present in green coffee. Weckerle et al.15 found only 3(S)- linalyl-3-O-β-D-glucopyranosyl-β-D-apiofuranoside and 3(S)-linalyl-3-O-β-D-glucopyranosyl-β-L-apiofuranoside, but not the corresponding R-forms. Selmar et al.16 pro- posed that during dry processing, coffee seeds have a more active metabolism because the slower drying initiates the seed germination. They further stated that the progression of the related metabolism depends on the mode of processing.
To gain information about sensory influences of the enantiomeric distribution, we determined the flavour recognition thresholds of the two stereoisomers in water and in espresso coffee from a commercial Arabica blend.
For water we found approximately 10 times the detection threshold published by Padrayuttawat et al.6 resulting in a recognition threshold of 95 ppb in water for S-linalool and 22 ppb for R-linalool.
In espresso coffee the corresponding BET levels were 6.84 mg/l and 0.93 mg/l for S- and R-linalool, respec- tively. The descriptive profile of the samples indicated that R-linalool elicits honey, flowery and caramel notes in espresso coffee. The intensity of the perception increases with increasing concentration. The sensory behaviour of the S-isomer is ambiguous: in low concentration it elicits a woody note, which disappears with the increas- ing concentration of the compound and is substituted by an intense flowery note (see Table 2).
Linalool is known to be among the key aroma com- pounds in green and roasted coffee.18–20 The flavour of racemic linalool is reported to be floral, green bergamot and woody.21 Synergistic effects, especially in the dense and concentrated espresso preparation, could be respon- sible for the different impressions of the isomers.
Currently further experimental samples of the same plants from other origins are under evaluation to confirm elevated (S)-linalool levels in sun-dried coffees.
Acknowledgements—Thanks to the sensory panel of Illycaffè for the endless sessions and Dr M. Schwarz for continuous discussion and as- sistance with the manuscript.
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