CURRENT MICROBIOLOGY Vol. 42 (2001), pp.

252–256
DOI: 10.1007/s002840110213 Current
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2001

Microbiological and Biochemical Study of Coffee Fermentation

Sylvie Avallone,1 Bernard Guyot,2 Jean-Marc Brillouet,3 Eugenia Olguin,4 Joseph-Pierre Guiraud1
1
USTL F-34095, GBSA-MBI CC 23 Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
2
CIRAD-CP, Montpellier Cedex, France
3
CIRAD-FLHOR, Montpellier Cedex, France
4
Instituto de Ecologia, Xalapa, Veracruz, Mexico

Received: 21 August 2000 / Accepted: 25 September 2000

Abstract. The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative
bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was
observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin
medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged
mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous.
The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola
and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation,
60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic
microorganisms.

Coffee preparation requires the elimination of the enve-
lopes from the beans and can be carried out by a dry or
wet process. In the wet method, coffee cherries are first
depulped in order to remove the skin (exocarp) and the
pulp (outer mesocarp). Beans are then put in a fermen-
tation tank with a water stream and allowed to ferment to
degrade a hygroscopic mucilaginous layer (inner meso-
carp) which constitutes an obstacle to the drying. The
quantity of water used in the wet process can be dimin-
ished by using dry pulping and water recycling [16, 26].
During the natural fermentation, the mucilaginous
substrate, rich in simple sugars and pectin, is liquefied [9,
11, 15, 17]. Coffee fermentation microflora are consti-
tuted by the classical microorganisms of vegetables,
fruits, and the hearth surrounding the trees [1, 14, 33].
The most frequent lactic acid bacteria isolated are Leu-
conostoc, Lactobacillus plantarum, and Lactobacillus
brevis [24, 32]. Yeasts have also been detected, but their
role in mucilage degradation is controversial [1, 14, 30].
The pectic substances would be degraded by the com-
bined action of microbial and/or endogenous coffee en-
zymes [2, 7, 13, 22, 32], but no biochemical proof was
ever done. Mucilage degradation is generally attributed
to pectolytic microorganisms, but further studies have
Correspondence to: J.P. Guiraud; email: guiraud@arpb.univ-montp2.fr.
demonstrated that the most frequent pectolytic strains,
Erwinia dissolvens, E. herbicola, and Klebsiella pneu-
moniae [4, 31], produce a pectatelyase unable to depo-
lymerize highly methylated pectins of coffee mucilage
[5, 10].
Until now, the evolution of each microflora (aerobic,
anaerobic, lactic, yeast, and pectolytic microflora) was
not studied as well as the biochemistry of the fermenta-
tion. The purpose of our work was to describe, quanti-
tatively and qualitatively, the microbial and biochemical
parameters of coffee fermentation in order to better un-
derstand the role of microorganisms in mucilage degra-
dation.
Materials and Methods
Plant material and processing. Coffee cherries (Coffea arabica L.
var. typica Cramer) were hand-picked at the mature stage in a planta-
tion from the Coatepec area (Xalapa, Veracruz, Mexico) during the
1998 year. External mesocarp (skin and pulp) was mechanically elim-
inated by wet or dry pulping (DV-225C PENAGOS depulper).
Depulped beans were then conveyed in a water stream to tanks and left
to ferment for 20 h. Every 5 h, samples were withdrawn from three
different locations at the middle depth of each tank and mixed.
pH, temperature and dry weight determinations. Fermented beans
(20 g) were gently swirled in distilled water, and the pH of the
supernatant was measured [20]. Temperature of the fermenting coffee
was determined with a portable electronic thermometer. Dry weight
S. Avallone et al.: Coffee Fermentation
was evaluated on samples left 3 h at 50°C and overnight at 60°C in a
vacuum oven.
Enumeration media. Fermented beans (20 g) were homogenized with
a Waring blender in bacto-NaCl solution (180 ml) during 1 min, and
decimal dilutions were then prepared in 8 g/l sterile sodium chloride.
Lactic microflora were counted on De Man, Rogosa and Sharpe agar
(MRS) [12], and yeast on Yeast Glucose Chloramphenicol agar (YGC)
containing 250 mg/l of antibiotic. Aerobic microorganisms were num-
bered on Plate Count Agar (PCA), and anaerobic bacteria on Meat-
Liver S.R. agar with incubation in anaerobic tanks. Microorganisms
able to grow with apple pectin (Sigma) as the sole source of carbon
were enumerated on Boccas medium, pH 6.8 [6]. After 2 days of
incubation at 30°C, colony-forming units (CFU) were counted in
duplicate, and data were expressed as the mean of the decimal loga-
rithm of the CFU/g of depulped bean dry weight. The most frequent
strains were isolated and stored at ⫺80°C in 30% glycerol. Strain
frequencies were calculated as the ratio of the respective population
density. All media used in our experiments were purchased from
Biokar Diagnostics company (France) except that of Boccas. Identifi-
cations were performed with API systems (BioMérieux, France), and
data were analyzed with an APILAB program calculating the most
probable bacteria.
Biochemical analysis. Simple sugars were extracted twice at room
temperature from whole pulped frozen beans with 80% ethanol for 1 h
under gentle stirring. Supernatants were pooled, concentrated, and the
volume was adjusted to 200 ml. The sugars were analyzed by high-
performance anion-exchange chromatography with a Dionex DX-300
system equipped with a PAD II detector and a Carbopac PA-1 column
(4 ⫻ 250 mm) with a Carbopac PA-1 guard column (4 ⫻ 50 mm). The
elution was performed at 1 ml.min⫺1 with 0.026 M NaOH and post-
column addition of 0.3 M NaOH. Organic acids were extracted at room
temperature from 5 g of pulped frozen beans with 5 mM H2SO4 (20 ml)
for 1 h. The solution was centrifuged and filtered on cellulose nitrate
(0.2 ␮m). Organic acids were then separated on an Applied Biosystems
Polypore H PPH-257 column (250 ⫻ 7 mm) eluted with 5 mM H2SO4
at 0.3 ml.min⫺1 [27]. Detection was achieved with a RID-6A Shimadzu
refractometer. Injected volume was 20 ␮l. Ethanol was measured by
gas-liquid chromatography on a packed Porapak Q column (1 m ⫻ 2.2
mm; 80 –100 mesh) at 170°C with nitrogen as carrier gas (20
ml.min⫺1). Injector and detector temperature was 200°C, and 2-propa-
nol was used as the internal standard.
253
Fig. 1. Physicochemical parameters of coffee fer-
mentation.
Results
Evolution of physical parameters. At the beginning of
the fermentation, the dry weight of depulped beans was
47%. The time necessary to decompose the mucilage
layer was approximately 20 h. During the first 10 h,
which corresponds to the night, the pH decreased slowly
because the outside temperature was rather cold (13°C)
(Fig. 1). Then acidification due to the microflora metab-
olism was observed. Coffee temperature variations were
moderate, but mesophilic microflora maintained a higher
temperature in tanks than outside during all the fermen-
tation step.
Quantitative study of coffee fermentation microflora.
The microbial counts are reported on a logarithmic scale
in Fig. 2. The initial wild microflora were rich and
remained constant during the first 10 h. This lag time was
due to the low temperature of the night and the time for
microorganisms to adapt their metabolisms. Between 10
and 15 h, the total microflora increased from 2.5 ⫻ 107
to 13.5 ⫻ 107 UFC/ml. The aerobic mesophilic micro-
flora were the quantitatively most important and dimin-
ished after 20 h because of low pH. Of the total aerobic
initial microflora, 0.1% grew on a medium with apple
pectin as the sole source of carbon, but no rise was
observed during the process.
Qualitative study of coffee fermentation microflora.
Aerobic bacteria. The evolution of the most numerous
strains is shown in Table 1. Most of the aerobic bacteria
were Gram-negative bacilli. The strains isolated with the
highest frequencies were coliforms commonly distrib-
uted in the environment and identified as Klebsiella
(73%) and Erwinia (28%). Bacteria having a respiratory
and fermentative metabolism with the production of a
low level of organic acids (Erwinia, Aeromonas) were