Eur Food Res Technol (2005) 220:245–250DOI 10.1007/s00217-004-1033-z

O R I G I N A L P A P E R

Gerhard Bytof · Sven-Erik Knopp · Peter Schieberle ·Ingo Teutsch · Dirk Selmar

Influence of processing on the generationof g-aminobutyric acid in green coffee beans

Received: 16 July 2004 / Revised: 19 August 2004 / Published online: 9 November 2004� Springer-Verlag 2004

Abstract A determination of the concentrations of freeamino acids in differently processed green coffees indi-cated the nonprotein amino acid g-aminobutyric acid(GABA), a well-known plant stress metabolite, to bepresent in raw coffee beans (Coffea arabica L.) in sig-nificantly varying amounts. The GABA content of un-washed Arabica beans (green coffee produced by the dryprocessing method) was always markedly higher than thatof washed Arabicas (wet processing method) as well asthat of untreated seeds. This result underlined the as-sumption that during postharvest treatment a significantmetabolism occurs within coffee seeds. A putative rela-tion between drought stress of the coffee seeds andpostharvest treatment methods is discussed. The GABAcontent of green coffee beans may serve as a potent toolto characterize the type of postharvest treatment appliedin coffee processing.

Keywords Coffea arabica · g-Aminobutyric acid · Greencoffee · Postharvest treatment · Coffee processing

Introduction

Traditionally, green coffee (Coffea arabica L.) is pro-duced by two different methods, known as wet and dryprocessing. Both methods are aimed at removing the fruitflesh of the coffee cherry and reducing the water contentin the raw coffee beans to about 10–12%. It is well ac-cepted that green coffee resulting from either method oftreatment, referred to as “washed” and “unwashed” Ara-

bicas, later on yields roasted beans and coffee beveragesclearly differing in aroma and taste [1]. These flavourdifferences are mostly attributed to differences in thethoroughness applied during either method of postharvesttreatment [2, 3].

The wet procedure requires a strict sorting of the fruitsprior to processing, because this treatment involves amechanical depulping step, which can only be executedon fully ripe coffee cherries. The dry processing method,however, can also be applied to unripe and overripe fruits,since this approach involves just a simple drying andhulling of the coffee fruits.

There is no doubt that factors such as the grade and thehomogeneity of the material affect the quality of the endproduct. However, according to our previous results [4]these appear to play a minor role, because we demon-strated that the typical differences in cup quality betweenwashed and unwashed Arabicas also occurred whenidentical and thoroughly sorted starting material was usedfor both methods of coffee processing. Correspondingdata had been published earlier [5, 6, 7, 8, 9]; however,previously biochemical reactions occurring in the seedshad never been taken into account. Apart from the sensorydifferences, we proved that the wet and dry processedsamples also differed significantly in the quality andquantity of free amino acids [4, 10]. Furthermore, specificand highly dynamic changes depending on the processwere detected. In previous studies we showed that thetotal content of free protein-amino acids is markedlyhigher in wet-processed coffee seeds than in dry-pro-cessed beans [4]. Later, it turned out that these differenceswere mainly due to a higher content of glutamic acid inwet-processed coffee seeds in comparison to dry-pro-cessed beans [10]. Previously, we had postulated that suchchanges in the chemical composition were related todifferences in the induction of germination [4, 10, 11].Therefore, the purpose of this study was to analyse thechanges induced in single amino acid concentrationsduring processing in more detail.

G. Bytof · S.-E. Knopp · D. Selmar ())Institute of Plant Biology,Technical University Braunschweig,Braunschweig, Germanye-mail: d.selmar@tu-bs.deTel.: +49-531-3915881Fax: +49-531-3918180

P. Schieberle · I. TeutschGerman Research Center for Food Chemistry (DFA),Munich, Germany

Material and methods

Batch processing

The field experiments were carried out in June and July 2002. InBrazil, fruits of C. arabica var. Acai� were processed at the fa-cilities of Ipanema Agricola, Alfenas, Minas Gerais, Brazil. Thebatch processings in Tanzania were performed using the C. arabicavar. KP 345 at the facilities of Tchibo estate, Moshi, Tanzania. Forthe experiments, only fully ripe and sound coffee fruits were used.This was ensured by thorough manual sorting of single batches offreshly harvested coffee cherries.

Wet processing

In Brazil, the coffee fruits were mechanically depulped using aPinhalense drum pulper, and submitted to 22 h of underwater tankfermentation. Subsequently, the washed parchment coffee beanswere dried on a separate plot of the sun terrace of the Ipanemafactory Conquista. After 6 days, a water content of 12% (wet basis)was achieved. In Tanzania, a McKinnon disk pulper was used.Coffee tank fermentation was performed as for dry fermentation, ina drained tank for 40 h. The washed parchment coffee was dried onseparate racks on drying tables in the sun until a water content of12% (wet basis) was reached.

Dry processing

In Brazil, the coffee cherries from the same batch used for the wetprocessing (see earlier) were dried as whole fruits on a separate plotof the sun terrace (final water content 12%). In Tanzania, the dryprocess is usually not applied and, therefore, the Tanzanian dry-processed coffee resulted exclusively from experimental process-ings by sun-drying of the coffee cherries on a patio. However,owing to the humid climate in Tanzania, a water content of onlyabout 18% was achieved. To complete drying, the seeds were driedat 35 �C in an oven in Germany.

Shipping of the coffee samples

Within 4 weeks after field processing, the dry parchment coffeesand the dry coffee cherries were sent to Germany and were man-ually dehulled and dehusked, respectively, and subsequently frozenin liquid nitrogen. The samples were stored at �70 �C prior to theanalysis.

Model processing

The fresh fruits of C. arabica were harvested on coffee plantationsin Tanzania and Mexico, respectively, and directly transferred toGermany by cargo express flights. To prevent the fruits from de-composing during transport, some plastic bags with ordinary icecubes were added. The cultivars used were C. arabica var. KP 345(Tanzania) and Caturra� (Mexico). Thorough manual sorting en-sured that only fully ripe and sound coffee cherries were used. Oneday after harvest, the fruits arrived in Germany and were imme-diately submitted to model processing.

Wet processing

For the model wet processing, the fruits were manually depulpedand the mucilaginous parchment beans were transferred into 5-LErlenmeyer-flasks and an excess of fresh water was added. Thecoffee was fermented at 21 �C for 36 h and the water was ex-changed three times. The resulting parchment coffee was dried in alaboratory oven at 35–40 �C until a water content of 12% (wet

basis) was achieved. The beans were manually dehulled and storedat �70 �C prior to analysis.

Dry processing

For the model dry processing, mature coffee cherries were dried inan oven at 35–40 �C until a water content of 12% (wet basis) wasachieved. The beans were manually dehusked and stored at �70 �Cprior to analysis.

Extraction and determination of free amino acids

To exclude variabilities due to individual variations, 60 sound andfaultless beans were weighed and pooled as one sample, frozenwith liquid nitrogen and ground to a fine powder using a RetschMM 200 ball mill. An aliquot (500 mg) was used for extraction.After adding norvaline (0.8 �mol per sample) as the internal stan-dard, the powder was repeatedly extracted with sulphosalicylic acid(4% w/v). The extracts were adjusted to 100 mL, centrifuged andfiltered.

The amino acids were either derivatized with o-phthaldialde-hyde (OPA) prior to high-performance liquid chromatography(HPLC) analysis or separated on an ion-exchange column withpostcolumn detection using ninhydrine. The OPA-derivatizationprocedure was performed according to the method in Ref. [12];however, a Spark Holland Midas autosampler was used for deriv-atization and sample injection. The derivatives were separated on aC18 column (Nucleosil 100, 5 �m, Macherey & Nagel,250�4.0 mm) using a binary gradient (A: 5% MeOH, 5% aceto-nitrile, 2% tetrahydrofuran, 88% 50 mM sodium acetate buffer,pH 6.2, B: 40% MeOH, 40% acetonitrile, 20% sodium acetatebuffer) at a flow rate of 1.3 mL/min. The derivatives were detectedby means of an RF-551 Shimadzu fluorescence detector(lex=334 nm; lem=425 nm) and quantified by external calibration.The analysis of free amino acids was accomplished using an au-tomatic amino acid analyser (Biotronic LC3000), by means ofpostcolumn derivatization with ninhydrin. Data given as nanomolesper seeds correspond to the amino acid content per average coffeebean, calculated as the 60th part of the entire sample (see earlier).Compared with the often-used weight-per-weight basis, this pro-cedure allows not only the better detection of the molar shift ofamino acids, but also permits reliable comparisons of coffee beansexhibiting quite different water contents (i.e. 11% for processedseeds and about 45% in the case of fresh seeds).

Results

Because the content of free amino acids may showmarked variations among batches of green coffee [4], allexperiments were done on identical starting material.

In a previous study [4], in addition to the protein aminoacids a further amino compound was detected in theHPLC chromatograms. This compound was present invery high concentrations in dry-processed green coffeebeans, but only in minor amounts in wet-processed sam-ples (Fig. 1). Referring to earlier reports on the occur-rence of the nonprotein amino acid g-aminobutyric acid(GABA) in coffee seeds (see Discussion), we analysed ifthe compound in question might be GABA. Using variousHPLC-methods and derivatization procedures (OPA,ninhydrin) the corresponding amino acid was undoubt-edly identified as GABA.

In Fig. 2a, the concentrations of free amino acids in theuntreated, the wet-processed and the dry-processed beans

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of same batch are contrasted. In the untreated beans, theconcentrations of glutamic and aspartic acid were highest,followed by alanine and asparagine. The wet process ledto an increase in the concentration of glutamic acid,whereas the concentrations of aspartic acid, alanine andasparagine decreased. During the dry process, in partic-ular, the concentration of GABA increased, whereas theconcentrations of most of the other amino acids wereeither lower compared with the concentrations in theunfermented beans or were present in similar concentra-tions.

In the wet-processed Tanzanian batch sample (Fig. 2b)the concentration of glutamic acid was also highest, whilein the dry-processed beans GABA reached by far thehighest concentrations. Both model studies, simulatingthe coffee processing, the Mexico (Fig. 2c) and theTanzania processes (Fig. 2d) confirmed the data obtainedfor the industrially processed samples and showed veryhigh concentrations of GABA only in the dry-processedsamples. Glutamic acid, which was the most prominentamino acid in coffee seeds, behaved conversely. Thisindicates that the GABA accumulated in dry-processedseeds is derived from glutamic acid by decarboxylation –a well-known reaction in plant tissues [13, 14, 15].

Further trials were, therefore, performed with specialemphasis on the relation between the GABA and glutamicacid concentrations of wet- and dry-processed coffeebeans. In order to avoid difficulties in evaluation due tothe fact that different batches of green coffees varystrongly in their overall content of amino acids, the dif-

ferences detected are mentioned as molar ratios (Table 1).In Tanzania, the wet-processed coffee beans throughoutare dried correctly. The data mentioned as “faultily dried”resulted from an experimental field processing in whichthe drying time was extended deliberately. Moreover, inTanzania, the dry process is generally not applied. Thedata mentioned as “faultily dried” resulted exclusivelyfrom experimental field processings.

It is evident that GABA is accumulated when coffeeseeds are processed by the dry method. However, in somecases, when drying of wet-processed coffee is performedtoo slowly, high amounts of GABA were accumulated,too (Table 1); yet the corresponding values did not reachthose of the dry-processed beans. The overall highestGABA-to-glutamate ratio was detected in dry-processedbeans resulting from a drying procedure which, owing torainfalls, was accidentally extended.

The finding that GABA is accumulated in coffee beansduring dry processing allows the chemical differentiationof differently processed green coffees. However, the datado have much greater relevance for the general under-standing of coffee seed physiology and related physio-logical and biochemical processes occurring within seedsduring postharvest treatments. The accumulation ofGABA clearly shows that various metabolic reactionstake place in the coffee seeds while these are processed.The results confirm our hypothesis [4] that the metabo-lism is active during the whole treatment and that theextent of these metabolic processes strongly depends onthe processing conditions.

Fig. 1 High-performance liquidchromatography chro-matograms of o-phthaldialde-hyde derivatives of free aminoacids isolated from wet- anddry-processed raw coffee beans.The arrows indicate the posi-tion of g-aminobutyric acid.

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Discussion

The presence of various amounts of GABA in greencoffee beans had already been reported in previous studies[16, 17, 18, 19, 20], and Trautwein [21] recognized areciprocal relation between the concentrations of GABAand free glutamic acid in commercial green coffee sam-ples; however, no suggestions were made with respect to

the reasons of this phenomenon. This was due to a lack ofinformation, whether the coffees analysed had been pro-cessed using the wet or the dry method. Arnold andLudwig [22] analysed free amino acids in green coffeebeans of well-known sources as well as samples fromlaboratory model-processing experiments. Unfortunately,their effort of mimicking the dry processing did not reallyreflect the conditions in the field, but rather corresponded

Fig. 2 Amino acid composition of differentially processed greencoffees from various origins. a, b Samples processed in Brazil orTanzania according to the industrial procedures (batch process-ings). c, d Samples processed in the laboratory mimicking the wet

and dry processings, respectively (model-processings). Each valueis based on 3–6 independent procedures. b In Tanzania, the dryprocess is normally not applied. The data resulted exclusively fromexperimental field processings.

Table 1 Comparison of the concentrations of g-aminobutyric acid (GABA) and glutamic acid in differently processed green coffees fromvarious origins. A, B and C correspond to different lots of Tanzanian green coffee.

GABA (nmol/seed) Glutamate (nmol/seed) Molar ratio of GABAto glutamate

Fresh seeds Tanzania A 310 3,887 0.08Brazil 30 2,018 0.02Tanzania B 62 2,932 0.02Mexico 37 1,733 0.02

Wet Batch, Tanzania A 96 1,580 0.06Batch, Brazil 93 2,547 0.04Model, Tanzania B 140 3,430 0.04Model, Mexico 89 2,314 0.04

Dry Batch, Brazil 1,009 1,937 0.52Model, Tanzania B 1,860 2,470 0.75Model, Mexico 1,411 909 1.55

Wet (faultily dried) Batch, Tanzania A 264 2,196 0.19Dry (faultily dried) Batch, Tanzania A 2619 3,88 6.75

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to an intermediate way of processing, generally referredto as “semidry processing”. Moreover, the authors [22]used the 9-fluorenylmethylchlorofomate (FMOC-Cl)method —a technique widely recommended for aminoacid analyses [23]—which, however, failed to producereliable data for GABA–FMOC derivatives [24]. Conse-quently, GABA was not considered in these investiga-tions. Thus, despite the numerous studies on amino acidconcentrations in green coffees published so far, our datashow for the first time that GABA is accumulated only indry-processed green coffee beans. This finding also has atremendous impact on the general understanding of coffeebiochemistry. It clearly demonstrates that coffee seedssubmitted to processing do have an active metabolism,which is undoubtedly relevant for coffee quality. Whilefirst proofs for the occurrence of germination in processedcoffee beans are under study, the accumulation of thestress metabolite GABA in dry-processed coffees con-firms the idea that the metabolic state of the seeds clearlydepends on the conditions applied during coffee pro-cessing.

For many different plant tissues, GABA is described asbeing accumulated as a metabolite in response to variousstress conditions [25]. A strong decrease in water poten-tial—as it occurs during the drying of the coffee beans—corresponds to a classical water stress situation. Aprominent reaction of plant cells in response to waterstress is the accumulation of various compounds en-hancing the osmotic potential of the cells, for example,the amino acid proline [26]; however, a pronounced ac-cumulation of proline in coffee seeds was not observed[10].

In plants, GABA is reported to be formed through a-decarboxylation of glutamic acid, catalysed by a pyri-doxal 50-phosphate-dependent glutamate decarboxylase[15]. The enzyme is also calmodulin-dependent, whichimplies a rapid activation due to increased Ca2+-concen-trations in the cytosol [13, 14, 15]. GABA is furthermetabolized by the so-called GABA-shunt, a bypass ofthe Krebs-cycle [13, 14, 15]. Assuming that GABA ac-cumulation in coffee seeds also corresponds to a stressreaction, the question arises why this stress metabolite isonly accumulated in dry-processed seeds, although thewet-processed seeds were also dried after fermentation.However, it has to be noted that the time courses of dryingare quite different.

In this context, two events are of special interest:firstly, the moment when the extent of drying causes thestress response, and secondly, the time when the watercontent has decreased so much that the entire metabolismis largely shut down (to be expected below 25%). Con-sequently, the stress metabolite GABA can only be syn-thesized and accumulated within this time frame. Duringa traditional dry coffee processing in the field, the drying(from 50 to 12%) takes about 2–3 weeks, and the timeframe for the stress reaction thus comprises several daysup to 1 week. In contrast, the entire drying of wet-pro-cessed seeds lasts only 3–6 days and the time from theonset of stress response to metabolic shutdown may only

be 1 day or even less. Thus, only during the dry-pro-cessing procedure, the time for synthesis and accumula-tion of GABA is obviously sufficient to accumulatemarked amounts of this stress metabolite. These facts donot only explain the observed differences in the GABAcontent of wet- and dry-processed green coffees, but alsothe occurrence of GABA in wet-processed beans driedunder unfavourable conditions: when the climatic situa-tion does not allow rapid drying of the beans, the time inwhich GABA can be accumulated is increased (Table 1).

We suggest using the GABA content of green coffeesas a reliable indicator for the processing method applied,with low concentrations of GABA indicating the wet-processing procedure. These considerations corroboratethe suggestion of Casal et al. [20] to use glutamic acid(among others) as a possible tool for the statistical dis-crimination between green coffees subjected to differentpostharvest processes. However, our data show that onlythe combination of GABA and glutamic acid concentra-tions will result in reliable data.

Acknowledgements This research project was supported by theForschungskreis der Ern�hrungsindustrie (Bonn, Germany), theArbeitsgemeinschaft industrieller Forschungsvereinigungen and theMinistry of Economic Affairs (project no. AIF-FV 12181 and13588). The authors wish to thank the Neumann Group, KraftFoods, Ipamema Agricola, and Tchibo for financial and logisticsupport.

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