Page 8 - Shimadzu Journal vol.3 Issue2

Metabolomics

Discrimination of Kopi Luwak and regular coffee has been 2. Experimental
achieved using electronic nose data . However, selection of
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discriminant marker for authentication was not addressed. The Samples and chemicals
method currently employed by Kopi Luwak producers is by visual
Samples were divided into experimental and validation coffee sets.
and organoleptic testing. Both methods are inadequate because
The first set included twenty one coffee beans that were collected
visual examination would only be possible for green coffee bean
from several cultivation areas in Indonesia. Kopi Luwak and
prior to roasting. As for organoleptic testing, trained experts that
regular coffee samples of two species, Coffea arabica (Arabica)
could discriminate Kopi Luwak are very few and the test tends to
and Coffea canephora (Robusta), were utilized. Coffee samples
be highly subjective.
were obtained from 21 sampling points of three cultivation areas
in Indonesia (Java, Sumatera, Bali) as shown in Table 1. In the
Information flows in metabolic pathways are highly dynamic and
second experiment for GC-FID application, one Robusta civet
represent current biological state of individual cells. Hence,
coffee sample (Sample no. 17 in Table 1) is removed from analysis
metabolome has been considered as the best descriptor of
resulting in only 20 coffee bean samples. The experimental coffee
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physiological phenomenon . With this capability, metabolomics
sets include civet coffee (no. 1-6, Arabica) that had been digested
technique can be a powerful tool to elucidate variations in
by civet, and undigested beans referred to as regular coffees (no.
phenotype imposed by any perturbations such as gene
7-20, Arabica and Robusta). All coffee samples were treated
modification, environmental factor, and physical stress. Processes
identically for post harvesting. Coffee samples were roasted in
inside animal digestive tract could be translated as physical and
Probat-Werke von Gimborn Maschinenfabrik GmbH model BRZ 2
enzymatic stress to coffee bean, as it reported to pose smoother
(Probat, Rhein, Germany) at 205°C for 10 min and followed by
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surface and color changes after digestion . Thus, metabolomics
immediate air-cooling for 5 min. Roasted coffee beans were kept
technique was selected to seek and select discriminant marker for
in sealed Falcon tubes at -30°C until use.
authenticity assessment of Kopi Luwak. Metabolomics technique
The second set of coffee samples included validation coffee sets.
has been effectively applied to distinguish phytochemical
The first validation set consists of authentic Kopi Luwak,
compositions of agricultural products among different origins,
commercial Kopi Luwak, commercial regular coffee,
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varieties, and cultivars for quality control and breeding .
imitation/adulteration coffee, and blend coffee (Table 2a). The
second validation set consists of 3 civet coffees and 3 regular
In this study, gas chromatography coupled with quadrupole mass
coffees were bought commercially and 2 additional authentic civet
spectrometry (GC-Q/MS) based metabolic profiling was employed
coffees from the Indonesian Coffee and Cocoa Research Institute
to identify discriminant marker for differentiation of Kopi Luwak
(Table 2b). In addition, each civet coffee and regular coffee was
and regular coffee. A combination of gas chromatography and
mixed in equal proportions (50:50, wt %) to obtain representative
mass spectrometry (GC/MS) has demonstrated as an effective
coffee blends. A total of 17 coffee samples, 8 pure and 9 coffee
analytical platform as it provides high sensitivity, reproducibility
blends, were then analyzed by GC/MS to verify the established
and quantitation of large amount of metabolites within a single
protocol for coffee authentication. All coffee samples were
8-9
step extraction . Samples classification by means of
measured in triplicates. All chemicals used in this study were
chemometrics was performed using principal component analysis
analytical grade.
(PCA). Subsequently, orthogonal projection to latent structures
combined with discriminant analysis (OPLS-DA) and significance
analysis of microarrays/metabolites (SAM) to identify statistically Extraction and derivatization
significant compounds as discriminant marker candidates, was
utilized 10-11 . The applicability of discriminant marker candidates To produce fine powder for extraction, coffee beans were ground
was verified to determine authenticity of commercial coffee. with a Retsch ball mill (20 Hz, 3 min). Fifteen mg of coffee powder
was extracted with 1 mL MeOH/CHCl3/H2O (5/2/2) and added with
However, a major drawback of this GC-MS-based approach is the 60 µL of ribitol (0.2 mg/mL) as internal standard. The samples
high cost of the instrument and maintenance. Therefore, an were centrifuged at 16000 g for 3 min at 4°C. Nine hundred
alternative method is needed for quality and authenticity microliter of the supernatant was then transferred into 1.5 mL
evaluation of civet coffee. A rapid, reliable and cost-effective Eppendorf tube and added with 400 µL Milli-Q water. After
analysis employing a universal detector, GC coupled with flame re-centrifugation, 400 µL aqueous layer was transferred into a
ionization detector (FID), and metabolite fingerprinting has been new tube with a pierced cap. The extract was evaporated by
established for discrimination analysis of 37 commercial and vacuum centrifugation for 2 h and freeze-drying overnight. The
non-commercial coffee beans extracts. GC/FID provided higher dried extract was mixed with 100 µL of methoxyamine
sensitivity over a similar range of detected compounds than hydrochloride (20 mg/mL in pyridine) and subsequently incubated
GC/MS. In combination with multivariate analysis, GC/FID could at 30°C for 90 min. The second agent, 50 µL MSTFA was added to
successfully reproduce quality prediction from GC/MS for the mixture and re-incubated at 37°C for 90 min.
differentiation of commercial civet coffee, regular coffee and
coffee blend with 50 wt % civet coffee content without prior
metabolite details. Our study demonstrated that GC/FID-based
metabolite fingerprinting can be effectively actualized as an
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alternative method for coffee authenticity screening in industries .
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